Mixture of polymers, lubricating fluid and porous materials comprising said mixture, and surface bearing said mixture

ABSTRACT

A mixture of polymers with lubricating properties is provided. The polymer can be used to produce a lubricating fluid. They can also be born on a surface or embedded in a porous material. This mixture of polymers comprises (a) a pharmaceutically acceptable bottle-brush polymer comprising a backbone with polymeric pendant chains, and (b) a pharmaceutically acceptable linear polymer. In the lubricating fluid, the bottle-brush polymer and the linear polymer are dissolved together in a pharmaceutically acceptable solvent.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.16/095,051, filed Oct. 19, 2018, which is a National Entry applicationof PCT application no PCT/CA/2017/050461 filed on Apr. 13, 2017 andpublished in English under PCT Article 21(2), which itself claimsbenefit of U.S. provisional application Ser. No. 62/326,253, filed onApr. 22, 2016. All documents above are incorporated herein in theirentirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DMR1436219 awardedby the National Science Foundation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

The present invention relates to a mixture of polymers with lubricatingproperties. More specifically, the present invention is concerned with alubricating fluid and its use in providing lubrication at a desired siteof action in or on a living body; a lubricated surface bearing thepolymers, and a porous material having embedded therein said polymers.

BACKGROUND OF THE INVENTION

With the ever-increasing need of more efficient and long lastingmachinery and devices, certain issues such as control of wear andfatigue of machine parts have become extremely challenging. The designof lubricating fluids able to protect surfaces against wear and highfriction has been one the several tools used by engineers to improvemachines' lifetimes.

It is generally assumed that damage caused during sliding, commonlyknown as “abrasive friction”, is due to a high friction force and,therefore, a large coefficient of friction. Accordingly, to preventsurface damage or wear, one should aim to reduce the coefficient offriction, which has been the traditional focus of basic research intomany bio- and non-biolubrication systems. However, many biological andnonbiological systems (especially involving soft polymeric surfaces)exhibit very complex behavior where the coefficient of friction and wear(abrasion) are not simply related and sometimes even have an inverserelationship. Therefore, other actors, such as the surface structure,the lubricant distribution and conformation, and the lubricant—surfaceinteraction, are certainly more important than the coefficient offriction in determining the onset of wear.

Several diseases have a degenerative mechanical component, such asosteoarthritis (mechanical wear of joints), lacrimal fluid productiondeficiencies (dry eye syndrome), or vaginal dryness. Osteoarthritisoccurs when protective cartilage situated at the ends of bones wear downover time. This can damage any joint in the body, and frequently affectshands, hips, knees, and the spine. Dry eye syndrome occurs when there isan insufficient or sub-optimal production of tears, usually due to aninsufficient or sub-optimal production of lacrimal fluid. For each ofthe above diseases, the prior art discloses several treatments.

The prior art discloses that while no known cure exists forosteoarthritis, the pain can be reduced and joint movement can bemaintained using various treatment methods, including medications (suchas acetaminophen and nonsteroidal anti-inflammatory drugs), therapy(such as physical therapy and occupational therapy), and varioussurgical procedures (including joint replacement and bone realignment)and intra-articular injections (including cortisone shots and hyaluronicacid injections).

Various treatment methods for dry eye syndrome are known, includingprescription medications. These include drugs to reduce eyelidinflammation; eye drops to control cornea inflammation; eye inserts;tear-stimulating drugs; and eye drops made from a patient's own blood.The prior art also discloses other procedures, such as closing tearducts; using special contact lenses; unblocking oil glands; and usinglight therapy and eyelid massages.

Various treatment methods for vaginal dryness are also known, includinglubricants and medication.

The prior art discloses hyaluronic acid and its use in variousbiomedical applications. Specifically, the use of hyaluronic acid totreat the above degenerative diseases is known, due to itsanti-inflammatory as well as its chondroprotective qualities. Forexample, the prior art discloses that osteoarthritis may be treated byinjecting hyaluronic acid into the joint where it increases theviscosity of synovial fluid and tempers inflammation processes. Inaddition, the prior art discloses that hyaluronic acid can be used ineye drops to treat dry eyes, as hyaluronic acid is found in the vitreousfluid of the eyes. Further, the prior art discloses that hyaluronic acidcan be used to make a lubricant gel used in the treatment of vaginaldryness.

Turning now to another topic, bottle-brush polymers are also known, andsuch polymers are known to have various applications.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided:

-   1. A lubricating fluid comprising:    -   a. a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b. a linear polymer,    -   the bottle-brush polymer and the linear polymer being dissolved        together in a solvent.-   2. The lubricating fluid according to item 1, wherein the backbone    of the bottle-brush polymer is acrylate or methacrylate based.-   3. The lubricating fluid according to item 1 or 2, wherein the    backbone of the bottle-brush polymer is poly(methacrylate) or a    poly(alkyl methacrylate).-   4. The lubricating fluid according to any one of items 1 to 3,    wherein the backbone of the bottle-brush polymer is poly(methyl    methacrylate).-   5. The lubricating fluid according to any one of items 1 to 4,    wherein the polymer in the pendant chains is attached directly to    the backbone of the bottle-brush polymer or attached via a linking    group.-   6. The lubricating fluid according to item 5, wherein the polymer in    the pendant chains is attached to the backbone of the bottle-brush    polymer via the linking group.-   7. The lubricating fluid according to item 5 or 6, wherein the    linking group attaching the polymer in the pendant chains to the    backbone of the bottle-brush polymer is a carboxylic acid, an ester,    an amine, an azide, or a thiol functional group, or an alkylene,    alkenylene, or alkylene group that is interrupted or not with one or    more ester, amine, or azide, or thio functional group.-   8. The lubricating fluid according to any one of items 1 to 7,    wherein the pendant chains are zwitterionic.-   9. The lubricating fluid according to any one of items 1 to 8,    wherein the polymer in the pendant chains comprises poly(acrylic    acid), poly(alkyl acrylic acid), poly(methacrylate), or poly(alkyl    methacrylate).-   10. The lubricating fluid according to item 9, wherein the polymer    in the pendant chains comprises poly(methyl methacrylate).-   11. The lubricating fluid according to any one of items 1 to 10,    wherein the polymer in the pendant chains has attached thereto a    substituent either directly or through a linking group.-   12. The lubricating fluid according to item 11, wherein the    substituent is attached through the linking group.-   13. The lubricating fluid according to item 11 or 12, wherein the    linking group attaching the substituent to the polymer in the    pendant chains is a carboxylic acid, an ester, an amine, an azide,    or a thiol functional group, or an alkylene, alkenylene, or alkylene    group that is interrupted or not with one or more ester, amine, or    azide, or thiol functional group.-   14. The lubricating fluid according to any one of items 11 to 13,    wherein the substituent is a phosphorylcholine group, a saccharide    or disaccharide group including but not limited to glucose, sucrose,    lactose and their derivatives, such as D-gluconolactone and    lactobionolactone, or a biocompatible hydrophilic group such as    hydroxy, oligo(ethylene oxide), carboxy, amino, sulfo, thiol,    phosphate, or a derivative thereof.-   15. The lubricating fluid according to any one of items 11 to 14,    wherein the substituent is phosporylcholine.-   16. The lubricating fluid according to any one of items 1 to 15,    wherein the polymer in the pendant chains is    poly(2-methacryloyloxyethyl phosphorylcholine) of formula:

-   17. The lubricating fluid according to any one of items 1 to 16,    wherein the bottle-brush polymer is a copolymer comprising the    following two monomers:

preferably a copolymer of formula:

-   -   preferably with a grafting ratio between about 40% and about        60%, more preferably between about 45% and about 55%, and most        of about 45% or about 55%.

-   18. The lubricating fluid according to any one of items 1 to 17,    wherein the bottle-brush polymer is of formula:

-   -   preferably with a grafting ratio from about 40% to about 60%,        more preferably from about 45% to about 55%, and most preferably        of about 45% or about 55%.

-   19. The lubricating fluid according to any one of items 1 to 18,    wherein the bottle-brush polymer is    (PBiBEM₅₄₀-g-PMPC₂₈)-stat-PHEMA₆₀-stat-PMMA₆₀₀    (PBiBEM₄₅₆-g-PMPC₃₅)-stat-PHEMA₃-stat-PMMA₃₇₀.

-   20. The lubricating fluid according to any one of items 11 to 9,    wherein the polymer in the pendant chains comprises hyaluronic acid.

-   21. The lubricating fluid according to any one of items 11 to 14,    wherein the substituent is a saccharide group or disaccharide.

-   22. The lubricating fluid according to item 21, wherein the    substituent is glucose, sucrose, lactose, or a derivative thereof.

-   23. The lubricating fluid according to any one of items 1 to 22,    wherein the bottle-brush polymer further comprises one or more    capping blocks.

-   24. The lubricating fluid according to any one of items 11 to 23,    wherein the molecular weight of the backbone is about 10 kDa, about    20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa,    about 70 kDa, about 80 kDa, about 100 kDa, about 200 kDa, about 300    kDa, about 400 kDa, about 500 kDa, about 750 kDa, or about 900 kDa    or more and/or about 1000 kDa, about 750 kDa, about 500 kDa, about    400 kDa, about 300 kDa, about 200 kDa or about 100 kDa or less.

-   25. The lubricating fluid according to any one of items 1 to 24,    wherein the molecular weight of the backbone of the bottle-brush    polymer is about 88700 Da.

-   26. The lubricating fluid according to any one of items 1 to 25,    wherein the molecular weight of the pendant chain is about 1 kDa,    about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa,    about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, or about 90    kDa or more and/or about 100 kDa, about 90 kDa, about 80 kDa, about    70 kDa, about 60 kDa, about 50 kDa, about 40 kDa, about 30 kDa,    about 20 kDa, or about 15 kDa or less.

-   27. The lubricating fluid according to any one of items 1 to 26,    wherein the molecular weight of the pendant chain is about 13275 Da.

-   28. The lubricating fluid according to any one of items 1 to 27,    wherein a grafting ratio of the bottle-brush polymer is between    about 30 and about 100%.

-   29. The lubricating fluid according to any one of items 1 to 28,    wherein a grafting ratio of the bottle-brush polymer is about 30%,    about 35%, about 40%, about 45%, or about 50% or more and/or about    100%, about 90%, about 80%, about 70%, about 65%, about 60%, about    55%, or about 50% or less.

-   30. The lubricating fluid according to any one of items 1 to 29,    wherein the grafting ratio of the bottle-brush polymer is between    about 40 and about 60%, preferably between about 45% and about 55%,    most preferably is about 45% or about 55%.

-   31. The lubricating fluid according to any one of items 1 to 30,    wherein the concentration of the bottle-brush polymer in the    lubricating fluid is about 1, about 25, about 50, about 75, about    85, about 90, about 95, about 100, about 150, about 200, about 250,    about 300, about 350, about 400, about 450, or about 500 ug/ml or    more and/or about 10, about 5, about 1, about 0.5, about 0.25, or    about 0.1 mg/mL or less.

-   32. The lubricating fluid according to any one of items 1 to 31,    wherein the concentration of the bottle-brush polymer is 100 ug/mi    or about 350 ug/ml.

-   33. The lubricating fluid according to any one of items 1 to 32,    wherein the linear polymer is hyaluronic acid, dextran,    poly(vinylpyrrolidone), poly(ethylene glycol), hydroxypropyl    cellulose, a polymethacrylate polymer or copolymer, or a    polyacrylate polymer or copolymer, or a (preferably pharmaceutically    acceptable) salt thereof.

-   34. The lubricating fluid according to any one of items 1 to 33,    wherein the linear polymer is hyaluronic acid or    poly(vinylpyrrolidone), or a (preferably pharmaceutically    acceptable) salt thereof; preferably hyaluronic acid or a    (preferably pharmaceutically acceptable) salt thereof.

-   35. The lubricating fluid according any one of items 1 to 34,    wherein the linear polymer is partially crosslinked.

-   36. The lubricating fluid according to any one of items 1 to 35,    wherein the linear polymer has a molecular weight of about 5 kDa,    about 10 kDa, about 25 kDa, about 50 kDa, about 100 kDa, about 250    kDa, or about 500 kDa or more and/or 10 MDa, about 8 MDa, or about 5    MDa or less.

-   37. The lubricating fluid according to any one of items 1 to 36,    wherein the concentration of linear polymer in the lubricating fluid    is about 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1    mg/mL, 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, or about 5 mg/mL or    more, preferably about 0.01 mg/mL or more, more preferably about 0.1    mg/mL or more, yet more preferably about 1 mg/mL or more, and most    preferably about 0.9 mg/mL or about 2 mg/ml.

-   38. The lubricating fluid according to any one of items 1 to 37,    comprising the bottle-brush polymer and the linear polymer in a    bottle-brush polymer:linear polymer weight ratio between about 1:1    and about 1:20, preferably between about 1:2.5 and about 1:15, more    preferably between about 1:5 and about 1:10.

-   39. The lubricating fluid according to any one of items 1 to 38,    wherein the solvent is saline, preferably isotonic, preferably    buffered at a pH of about 7 to about 7.4.

-   40. The lubricating fluid according to any one of items 1 to 39,    wherein the solvent is phosphate-buffered saline (PBS).

-   41. The lubricating fluid according to any one of items 1 to 40,    further comprising one or more additive.

-   42. The lubricating fluid according to any one of items 1 to 41,    consisting of pharmaceutically acceptable ingredients only.

-   43. The lubricating fluid according to any one of items 1 to 42,    further comprising one or more therapeutic agent.

-   44. Use of a lubricating fluid as defined in any one of items 1 to    43 in providing lubrication at a desired site of action.

-   45. A method of method providing lubrication at a desired site of    action, the method comprising the step of applying a lubricating    fluid as defined in any one of items 1 to 43 at said site of action.

-   46. Use of a lubricating fluid as defined in item 42 or 43 in    providing lubrication at a desired site of action in or on a living    body e.g. of a human.

-   47. The use of item 46, wherein the desired site of action is an    eye, skin, a surface of a ligament, preferably a recently operated    ligament, a vagina, a joint, a gastrointestinal tract, a nasal duct,    a tracheal duct, or a stomach.

-   48. A method of lubricating a tissue of a living body e.g. of a    human, the method comprising the step of contacting the lubricating    fluid as recited in item 42 or 43 with said tissue.

-   49. The method of item 48, wherein the tissue is an eye, skin, a    surface of a ligament, preferably a recently operated ligament, a    vagina, a joint, a gastrointestinal tract, a nasal duct, a tracheal    duct, or a stomach.

-   50. Use of a lubricating fluid as defined in item 42 or 43 for the    treatment of a disease having a degenerative mechanical component.

-   51. The use of item 50, wherein the disease is osteoarthritis, a    lacrimal fluid production deficiency, or vaginal dryness.

-   52. A method of treating a disease having a degenerative mechanical    component, the method comprising administering a lubricating fluid    as defined in item 42 or 43 to a tissue affected by the disease.

-   53. The method of item 51, wherein the disease is osteoarthritis, a    lacrimal fluid production deficiency, or vaginal dryness.

-   54. The method of any one of items 48, 49, 52, and 53, wherein the    lubricating fluid is administered by injection.

-   55. The method of any one of items 48, 49, 52, and 53, wherein the    lubricating fluid is administered intravaginally.

-   56. The method of any one of items 48, 49, 52, and 53, wherein the    lubricating fluid is administered topically.

-   57. The method of anyone of items 48, 49, 52, and 53, wherein the    lubricating fluid is administered intranasally.

-   58. The method of any one of items 48, 49, 52, and 53, wherein the    lubricating fluid is administered orally.

-   59. A synthetic synovial fluid comprising a lubricating fluid as    defined in item 42 or 43.

-   60. The synthetic synovial fluid according to item 59, being for use    in the treatment of osteoarthritis.

-   61. Eye drops comprising a lubricating fluid as defined in item 42    or 43.

-   62. The eye drops according to item 61, being for use in the    treatment of dry eye.

-   63. A vaginal lubricating composition comprising a lubricating fluid    as defined in item 42 or 43.

-   64. The composition according to item 63, being for use in treating    vaginal dryness and/or infertility related to vaginal dryness.

-   65. Use of a lubricating fluid as recited in any one of items 1 to    43 in lubricating a medical instrument.

-   66. The use of item 65, wherein the medical instrument is a syringe,    an injection device, or an elution device

-   67. A method of lubricating of a surface of a medical instrument    comprising the step of contacting a lubricating fluid as defined in    any one of items 1 to 43 with said surface,

-   68. The method of item 67, wherein the medical instrument is a    syringe, an injection device, or an elution device

-   69. A mixture comprising:    -   a) a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b) a linear polymer,    -   wherein the bottle-brush polymer and the linear polymer are as        defined in any one of item 1 to 43.

-   70. The mixture of item 69, wherein the bottle-brush polymer and the    linear polymer are pharmaceutically acceptable.

-   71. The mixture of item 69 or 70, being in solid form, preferably in    the form of a powder.

-   72. Use of a mixture as defined in any one of items 69 to 71 for    producing the lubricating fluid of any one of item 1 to 43.

-   73. A method for producing the lubricating fluid of any one of item    1 to 43, the method comprising the step of contacting a mixture as    defined in any one of items 69 to 71 with a solvent, and allowing    dissolution of the mixture in the solvent.

-   74. The method of item 73, further comprising mixing the mixture    with the solvent to speed said dissolution.

-   75. The method of item 73 or 74, wherein the bottle-brush polymer,    the linear polymer, the solvent, and the lubricating fluid are    pharmaceutically acceptable.

-   76. The method of item 75, wherein, before said contacting step, the    mixture is administered to a subject, and wherein said dissolution    occurs in vivo, preferably at a site of action of the lubricating    fluid.

-   77. The method of item 76, wherein the solvent is a body fluid.

-   78. The method of item 77, wherein the mixture is administered    orally in the form of an oral formulation for releasing the linear    polymer and the bottle-brush polymer in a gastrointestinal tract,    and wherein, upon release, the linear polymer and the bottle-brush    polymer contact and dissolve in a fluid present in the    gastrointestinal tract.

-   79. The method of item 76, wherein the solvent is an extraneous    solvent.

-   80. A surface bearing a polymeric layer, the polymeric layer    comprising:    -   a) a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b) a linear polymer,    -   wherein the bottle-brush polymer and the linear polymer are as        defined in any one of item 1 to 43.

-   81. The surface of item 80, wherein the polymeric layer releases the    bottle-brush polymer and the linear polymer when the surface    contacts a solvent.

-   82. The surface of item 80, wherein the bottle-brush polymer and the    linear polymer remain in the polymeric layer on the surface for a    period of time when the surface is in contact with a solvent.

-   83. The surface of item 80 or 81, wherein said solvent is a body    fluid.

-   84. The surface of any one of items 80 to 83, wherein the polymeric    layer comprises the bottle-brush polymer and the linear polymer in a    bottle-brush polymer:linear polymer weight ratio between about 1:5    and about 1:15, preferably between about 1:7 and about 1:12, more    preferably between about 1:8 and about 1:9, most preferably of about    1:9.

-   85. The surface of any one of items 80 to 84, being a surface of an    ophthalmic lens, a surface of the barrel of a syringe, a surface of    an injection device, a surface of an elution device, or a surface of    an implant.

-   86. The surface of any one of items 80 to 86, wherein the    bottle-brush polymer and the linear polymer are pharmaceutically    acceptable.

-   87. A porous material having embedded therein:    -   a) a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b) a linear polymer,    -   wherein the bottle-brush polymer and the linear polymer are as        defined in any one of item 1 to 43.

-   88. The porous material of item 87, wherein the porous material is a    gel, a sponge, a textile or textile fibers.

-   89. The porous material of item 87 or 88, wherein the bottle-brush    polymer, the linear polymer, and the polymer material are    pharmaceutically acceptable.

-   90. The porous material of any one of items 87 to 89, wherein the    porous material comprises the bottle-brush polymer and the linear    polymer in a bottle-brush polymer:linear polymer weight ratio    between about 1:5 and about 1:15, preferably between about 1:7 and    about 1:12, more preferably between about 1:8 and about 1:9, most    preferably of about 1:9.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 shows the architecture of the bottle brush polymer.

FIG. 2 shows GPC traces recorded for B2 (solid line) and B2 MI (dashedline).

FIG. 3 shows the ¹H NMR spectra of B2 MI.

FIG. 4 shows synthetic pathways for the preparation of ABA bottle-brushcopolymers with PMPC (PMPC B2) pendant chains.

FIG. 5 shows the mechanical equivalent of the experimental surface forceapparatus (FSA) setup in the configuration used in Example 1.

FIGS. 6 A) and B) are AFM pictures of the bottle brush polymer on micain air.

FIG. 7 shows the contour length of the bottle-brush polymer.

FIG. 8 shows the interaction force profiles across different fluids asmeasured in the SFA.

FIG. 9 shows the interaction force profiles measured for HA solution (1mg/mL) at increasing ionic strength. Inset is an expanded view of theforce profile at 1500 mM NaCl showing characteristic step-likeinstabilities in the interaction forces, indicating the presence of alayered structure in the confined space.

FIG. 10 shows the interaction forces measured in presence of HA (1mg/mL) indifferent media. Out run (forces measured upon separation) arenot represented for clarity but presented systematically weak adhesion.

FIG. 11 shows the interaction force profiles measured for BB polymersolution (0.1 mg/mL) at increasing ionic strength.

FIG. 12 shows the interaction force profiles measured for a mixture ofBB polymer and HA 1500 kDa at different ionic strengths.

FIG. 13 shows the interaction forces between mica surfaces for mixturesof HA [A):1500 KDa, B) 500 kDa, C) 60 KDa, and D) 10 kDa] and BB polymerin pure water (top curves), in 150 mM NaCl (middle curves), and 1500 mMNaCl (top curves).

FIG. 14 is a schematic representation of the interfacial polymer layerin the presence of HA and BB polymers;

FIG. 15 shows a mixture of a bottle-brush polymer and a linear polymer.

FIG. 16 shows the tribological testing of the BB (0.1 mg/mL) and HA (1mg/mL) polymer mixtures in PBS (150 mM NaCl) performed at a slidingspeed of 3 μm/s.

FIG. 17 shows the shows measured friction coefficients in saline (150and 1500 mM NaCl) before and after damage in the presence of BB and HApolymers, alone and mixed together. Onset of damage is indicated by the“*” symbol.

FIG. 18 shows the film thickness and refractive index during shear(shearing speed v_(s)=3 μm/s). The film thickness D decreases graduallydue to the increase of the normal pressure/load during the course of theexperiment. When P═P*, damage of the surfaces occurs abruptly as shownby the rapid increase in D.

FIG. 19 shows the evolution of the critical pressure at which surfacedamage occurs (i.e. the thin film rupture pressure P*) under differentconditions, wherein HA concentration=1 mg/mL, BB polymer=0.1 mg/mL, andRatio HA/BB=10:1 at a sliding speed of 3 μm/s; and

FIG. 20 shows the thin film thickness during shear in pure water.

FIG. 21 shows the thin film thickness during shear in PBS.

FIG. 22 shows the differential power (top panel) and integrated releasedheat (lower panel) recorded during the titration of BB polymer into A)HA in buffered saline; B) buffered saline; and C) PVP in bufferedsaline.

FIG. 23 is a schematic representation of the wear protection mechanismobserved in the presence of the mixture of HA and BB polymers in purewater and in saline.

FIG. 24 shows the friction force in the presence of the BB polymer-HAmixture as a function of the sliding speed.

FIG. 25 shows A) the influence of the BB/HA (1.5 MDa) ratio on thecritical pressure, B) the influence of the nature of the linear polymer(PVP) on the critical pressure, and C) the influence of the surfaces(mica/gold) on the critical pressure.

FIG. 26 shows the cumulative dissipated energy generated during shearingof two 2.5 wt % chitosan hydrogel plugs, lubricated with differentpolymer solutions.

FIG. 27 shows interferometric micrographs of the hydrogel plugs after10⁴ shearing cycles at 5 mm/s (shearing amplitude of 5 mm, appliedpressure P=50 kPa): A) reference, B) PBS, C) HA, D) BB polymer, and E)polymer mixture.

FIG. 28 shows the surface roughness associated the interferometricmicrographs shown in FIG. 10.28.

FIG. 29 shows the Example of FECO fringes showing no damage (up) anddamaged appearance (down).

FIG. 30 shows the onset of interaction as a function of time atdifferent temperature.

FIG. 31 shows the kinetic constant as a function of 1/temperature.

FIG. 32 shows the steps of rat ACLT surgery as carried out in Example 2.

FIG. 33 shows the intra-articular injection into the rat knee.

FIG. 34 shows micrographs of the left femoral condyle (left column,control without surgery) and right femoral condyle (right column)treated with either HA or HA+BB together with the corresponding modulus.

FIG. 35 shows the loss of modulus with treatment with HA or HA+BB.

FIG. 36 shows micrographs of the left femoral condyle (left column,control without surgery) and right femoral condyle (right column)treated with either HA or HA+BB together with the corresponding meancartilage thickness.

FIG. 37 shows mean cartilage thickness variation with treatment with HAor HA+BB compared to CTL.

FIG. 38 shows the chemical structures of an exemplary bottle-brushpolymer and sodium hyaluronate.

DETAILED DESCRIPTION OF THE INVENTION

Lubricating Fluid

Turning now to the invention in more detail, there is provided alubricating fluid.

A lubricating fluid is a fluid provides lubrication at various desiredsites of action. In embodiments, the fluid is a pharmaceuticallubricating fluid, i.e. a lubricating fluid that is pharmaceuticallyacceptable and that can be used to provide lubrication at variousdesired sites of action, including in or on a living body, for examplein the eyes, vagina, or joints of e.g. a human.

The fluid may present different viscosities, from a watery consistencyto a gel-like consistency, depending on its end-use.

The lubricating fluid of the invention comprises:

-   -   a) a acceptable bottle-brush polymer comprising a polymeric        backbone with polymeric pendant chains, and    -   b) a acceptable linear polymer,        the bottle-brush polymer and the linear polymer being dissolved        together in a solvent.

In embodiments, either, some of or preferably all of the bottle-brushpolymer, the linear polymer, and the solvent are pharmaceuticallyacceptable.

The term “bottle-brush polymer” refers to a polymer comprising a linearpolymeric backbone with multiple polymeric pendant chains attached tothe backbone. FIG. 1 shows the typical architecture of a bottle-brushpolymer. The aforementioned backbone and pendant chains form the“bottle-brush” block in this polymer. Optional “capping blocks” locatedat either or both ends of the linear backbone of the “bottle-brush”block are also shown in FIG. 1.

The bottle-brush polymer can be characterized by its grafting ratio. Thegrafting ratio represents the percentage of repeat units of the backboneof the bottle-brush polymer that bear a polymeric pendant chain. For thebottle-brush polymer comprised in the present lubricating fluid, thegrafting ratio typically ranges between about 30 and about 100%. Inpreferred embodiments of the invention, the grafting ratio is about 30%,about 35%, about 40%, about 45%, or about 50% or more and/or about 100%,about 90%, about 80%, about 70% N, about 65%, about 60%, about 55%, orabout 50% or less. In most preferred embodiments, the grafting ratio isbetween about 40 and about 60%, preferably between about 40% and about55%, more preferably between about 40% and about 50%, yet morepreferably is about 45%.

The bottle-brush polymer can also be characterized by the molecularweight of its backbone and the molecular weight of its pendant chains.In embodiments, the molecular weight of the backbone is about 10 kDa,about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa,about 70 kDa, about 80 kDa, about 100 kDa, about 200 kDa, about 300 kDa,about 400 kDa, about 500 kDa, about 750 kDa, or about 900 kDa or moreand/or about 1000 kDa, about 750 kDa, about 500 kDa, about 400 kDa,about 300 kDa, about 200 kDa, or about 100 kDa or less. In preferredembodiments, the molecular weight of the backbone is about 90 kDa (e.g.88700 Da). In embodiments, the molecular weight of the pendant chain isabout 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa,about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, orabout 90 kDa or more and/or about 100 kDa, about 90 kDa, about 80 kDa,about 70 kDa, about 60 kDa, about 50 kDa, about 40 kDa, about 30 kDa,about 20 kDa, or about 15 kDa or less. In preferred embodiments, themolecular weight of the pendant chain is about 15 kDa (e.g. 13275 Da).

The bottle-brush polymer is also characterized by the nature of therepeat units in its backbone and the nature of the pendant chains.

The nature of the repeat units of the backbone of the bottle-brushpolymer is chosen so that it produces polymer (preferably apharmaceutically acceptable polymer) and allows for post-grafting of thependant chains. The grafting of the pendant chains may be achieved usingthe “grafted from” or “grafting to” methods. In the “grafted from”approach, the pendant chains are grown from a macromolecular chainbearing initiator functional groups. In the “grafting to” approach, thependant chains are polymerized separately and grafted to the main chainafterwards.

In embodiments, the backbone of the bottle-brush polymer may be acrylatebased (e.g. poly(acrylic acid), a poly(acrylate), such as poly(alkylacrylate) and more specifically poly(methyl acrylate)) or methacrylatebased (e.g. poly(methacrylic acid), a poly(methacrylate), such aspoly(alkyl methacrylate) and more specifically poly(methylmethacrylate)). In preferred embodiments, the backbone of thebottle-brush polymer is poly(methacrylic acid) or a poly(alkylmethacrylate), such as e.g. poly(methyl methacrylate).

As noted above, the pendant chains of the bottle-brush polymer arepolymeric. The polymer in these pendant chains may be attached directlyto the backbone of the bottle-brush polymer or attached via a linkinggroup.

Suitable linking groups (for attaching the polymer of the pendant chainto the backbone of the bottle-brush polymer) include carboxylic acid,ester, amine, azide, and thiol functional groups as well as alkylene,alkenylene, and alkynylene groups, the alkylene, alkenylene, andalkynylene groups being interrupted or not with one or more ester,amine, azide, and/or thiol functional groups. Herein “interrupted” meansthat a functional group (ester, amine, azide, or thiol) is located ateither end or in between two carbon atoms of the alkylene, alkenylene,and alkynylene groups.

In embodiments, the pendant chains are zwitterionic.

In embodiments, the polymer in the pendant chains is hyaluronic acid.

In other embodiments, the polymer in the pendant chains is poly(acrylicacid), a poly(acrylate)—such as a poly(alkyl acrylate) e.g. apoly(methyl acrylate—poly(methacrylic acid), a poly(methacrylate)—suchas a poly(alkyl methacrylate) e.g. poly(methyl methacrylate). Thesepolymer can optionally have attached thereto, directly or indirectlythrough a linking group, a substituent. Examples of such a substituentinclude phosphorylcholine

the open link attached to the oxygen atom on the left indicating thebond attaching the phosphorylcholine to the rest of the molecule),saccharide and disaccharide groups, including but not limited toglucose, sucrose, lactose and their derivatives such as D-gluconolactoneand lactobionolactone, as well biocompatible hydrophilic groups such ashydroxy, oligo(ethylene oxide), carboxy, amino, sulfo, thiol, phosphate,and derivatives thereof. Suitable linking groups (for attaching suchsubstituent to the polymer of the pendant chain) include carboxylicacid, ester, amine, azide, and thiol functional groups as well asalkylene, alkenylene, and alkynyene groups, the alkylene, alkenylene,and alkynylene groups being interrupted or not with or more ester,amine, azide, and/or thiol functional groups.

In preferred embodiments, the polymer in the pendant chains ispoly(methyl methacrylate).

In preferred embodiments, the polymer in the pendant chains has attachedthereto said substituent, preferably through said linking group.

In preferred embodiments, the substituent is phosphorylcholine.

In preferred embodiments, the polymer in the pendant chains ispoly(2-methacryloyloxyethyl phosphorylcholine of formula:

and in embodiments:

wherein x represents the number of repeat unit and, in embodiments,varies between 0 and 1000. In embodiments, x is preferably variesbetween 10 and 100, and more preferably between 10 and 50.

In embodiments, the bottle-brush polymer is a copolymer comprising thefollowing two monomers:

wherein x is as defined above. In embodiments, the copolymer has agrafting ratio, between about 40% and about 60% (in other words, itcomprises about 40% to about 60% of the monomer on the left), preferablyit has a grafting ratio between about 45% and about 55%, and morepreferably a grafting ratio of about 45% or about 55%.

In embodiments, the bottle-brush polymer is(PBiBEM-g-PMPC)-stat-PHEMA-stat-PMMA:

preferably

and more specifically:

preferably:

wherein x is as defined above and m and n represent repeat unit ratios(e.g. m=number of repeat units of the formula on the left/total numberof repeat units in polymer and as such vary between 0 and 1. Inembodiments, this bottle-brush polymer has a grafting ratio as describedabove, i.e. between about 30% and about 100% (i.e. m varies from about0.3 to about 1). In preferred embodiments, the grafting ratio is about30%, about 35%, about 40%, about 45%, or about 50% or more and/or about100%, about 90%, about 80%, about 70%, about 65%, about 60%, or about55% or about 50% less. In more preferred embodiments, the grafting ratiovaries from about 40% to about 60% (i.e. m varies from about 0.4 toabout 0.6), preferably about 45% to about 55% (i.e. m varies from about0.45 to about 0.55), more preferably about 40% to about 50% (i.e. mvaries from about 0.4 to about 0.5), and yet more preferably is about45% or about 55% (m is about 0.45 or about 0.55). In embodiments, thisbottle-brush polymer comprises a minor proportion of PHEMA, i.e. n isabout 0.15, about 0.10, about 0.08, about 0.05, about 0.04, about 0.03,about 0.02, about 0.01, about 0.0075, about 0.005, or about 0.004 orless, preferably n is about 0.05 or less; or is about 0.05 or about0.004. In preferred embodiments, the bottle brush polymer is of formula(PBiBEM₅₄₀-g-PMPC₂₈)-stat-PHEMA₆₀-stat-PMMA₆₀₀ or(PBiBEM₄₅₆-g-PMPC₃₅)-stat-PHEMA₃-stat-PMMA₃₇₀, wherein the numbersrepresent the number of repeat units in the polymer (i.e. in thesepolymers m=540/1200=0.45 and n=60/1200=0.05 and m=456/829=0.55 andn=3/829=0.0036, respectively).

In any of the above embodiments, the bottle-brush polymer may furthercomprise one or more aforementioned “capping blocks”. A capping block isa functional group, a substituent, or a polymer or peptide attached ateither or both ends of the backbone of the bottle-brush polymer. Thenature of capping blocks will be chosen according to the properties tobe imparted to the bottle-brush polymer. For example, capping blocksmight be included to improve adhesion of the bottle-brush polymer tobiological surfaces or biopolymers. Suitable capping blocks includealkyl, alkene or alkyne groups optionally bearing one or more thiol,amine, carboxylic, and/or azide functional groups, peptides, as well aspolymer chains bearing said functional groups or peptides.

In embodiments, the concentration of bottle-brush polymer is about 1,about 25, about 50, about 75, about 85, about 90, about 95, about 100,about 150, about 200, about 250, about 300, about 350, about 400, about450, or about 500 ug/ml or more and/or about 10, about 5, about 1, about0.5, about 0.25, or about 0.1 mg/mL or less. In preferred embodiments,the concentration of bottle-brush polymer is about 100 ug/ml (i.e. 0.1mg/mL) or about 350 ug/ml.

The linear polymer is a polymer that, contrary to the bottle-brushpolymer, has a linear structure. It is thus free from polymeric pendantchains.

In embodiments, the linear polymer has a molecular weight of about 5kDa, about 10 kDa, about 25 kDa, about 50 kDa, about 100 kDa, about 250kDa, or about 500 kDa or more and/or 10 Mda, about 8 Mda, or about 5 Mdaor less. In embodiments, the linear polymer has a molecular weight ofabout 1.5 Mda, about 500 kDa, about 50 kDa or about 10 kDa.

In embodiments, the linear polymer is:

-   -   hyaluronic acid:

-   -   dextran:

-   -   poly(vinylpyrrolidone):

or

-   -   poly(ethylene glycol):

-   -   hydroxypropyl cellulose,    -   a polymethacrylate polymer or copolymer, or    -   a polyacrylate polymer or copolymer,        wherein m and n represent the number of repeat units, or a        (preferably pharmaceutically acceptable) salt thereof. In        preferred embodiments, the number of repeat units is such that        the linear polymer has the abovementioned molecular weights. In        embodiments, the linear polymer is partially crosslinked to        achieve these molecular weights.

In preferred embodiments, the linear polymer is hyaluronic acid or a(preferably pharmaceutically acceptable salt) thereof, for examplesodium hyaluronate.

In other preferred embodiments, the linear polymer ispoly(vinylpyrrolidone).

In embodiments, the concentration of the linear polymer in thelubricating fluid is about 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05mg/mL, 0.1 mg/mL, 0.5 mg/mL, about 1 mg/mL, about 2 mg/mL, or about 5mg/mL or more. In preferred embodiments, the concentration of the linearpolymer in the lubricating fluid is about 0.01 mg/mL or more, and morepreferably about 0.1 mg/mL or more, yet more preferably about 1 mg/mL ormore, and most preferably about 0.9 mg/mL or about 2 mg/mL.

In embodiments, the lubricating fluid comprises the bottle-brush polymerand the linear polymer in a bottle-brush polymer:linear polymer weightratio between about 1:1 and about 1:20, preferably between about 1:2.5and about 1:15, more preferably between about 1:5 and about 1:10.

In embodiments, the bottle-brush polymer and the linear polymer arepresent in the lubricating fluid at a bottle-brush polymer:linearpolymer weight ratio between about

In embodiments, the solvent is saline (i.e. an aqueous solution ofmainly NaCl), preferably isotonic, preferably buffered at a pH of about7 to about 7.4 using for example a phosphate buffer. In embodiments, thesolvent is phosphate-buffered saline (PBS).

The lubricating fluid may further comprise one or more additive, such asfor example preservatives, colorants, flavorings, and odorants. Inembodiments, the additives are pharmaceutically acceptable.

Herein, “pharmaceutically acceptable” means generally accepted for usein pharmaceutical products. Of note, as is well know to the skilledperson, whether a product is pharmaceutically acceptable depends on theend use of the lubricating fluid. For example, components that may beacceptable in a fluid intended to be applied topically may not beacceptable when the fluid is intended to be administered by injection.

The lubricating fluid may further comprise one or more therapeuticagent. Such therapeutic agents may be chosen according to the end use ofthe lubricating fluid. For example, when the fluid is for application tothe eye, compounds know to be applied to the eye for treating variousconditions may also be incorporated in the fluid. In such embodiments,the lubricating fluid is preferably a pharmaceutical lubricating fluid,i.e. a lubricating fluid that consists of pharmaceutically acceptableingredients (i.e. polymers, solvents, additives, etc.) only.

Use and Properties of the Lubricating Fluid.

The present invention also provides the use of the above lubricatingfluid in providing lubrication at a desired site of action. There isalso provided a method of providing lubrication at a desired site ofaction comprising the step of applying the lubricating fluid at saidsite of action.

In embodiments, the site of action is in or on a living body of e.g. ahuman, for example the living body of a human. There is thus alsoprovided a method of lubricating a tissue of a living body of e.g. ahuman comprising the step of contacting the lubricating fluid with saidtissue. In such embodiments, the lubricating fluid preferably consistsof pharmaceutically acceptable ingredients (i.e. polymers, solvents,additives, etc.) only.

In embodiments, the site of action and/or tissue is the eye, the skin, asurface of a ligament, preferably a recently operated ligament, thevagina, a joint, the gastrointestinal tract, the nasal duct, thetracheal duct, or the stomach.

The use of this fluid is preferably for the treatment of diseases havinga degenerative mechanical component, such as osteoarthritis (mechanicalwear of joints), lacrimal fluid production deficiencies (dry eyesyndrome), or vaginal dryness.

The lubricating fluid may be administered in various ways according thecondition to be treated. For example, the lubricating fluid may beadministered:

-   -   by injection e.g. intra-particularly (in an articulation to be        treated),    -   intravaginally (in the vagina),    -   topically, for example to the eye (on the cornea), to the skin,        or to the surface of the ligament,    -   intranasally, for example, for treatment of the nasal duct or        the tracheal duct, or    -   orally, for example using a capsule, to lubricate the        gastrointestinal tract.

In an embodiment of the present invention, the lubricating fluid is usedfor the treatment of osteoarthritis by intra-articular injection. Thus,there is provided a synthetic synovial fluid comprising (or consistingof) the above lubricating fluid. In embodiments, the synthetic synovialfluid also comprises one or more pharmaceutically acceptable additiveand/or therapeutic agent as defined above.

In a further embodiment of the present invention, the lubricating fluidis used in the treatment of dry eye syndrome. Topical application ofthis fluid to the cornea is expected to allow for better retention ofwater on the surface of the eye and to reduce the adhesion between theeyelid and ocular epithelium (said adhesion being a source of pain),which commercial formulations currently cannot do. Thus, there areprovided eyes drops comprising (or consisting of) the above lubricatingfluid. In embodiments, the eyes drops also comprise one or morepharmaceutically acceptable additive and/or therapeutic agent as definedabove.

In a yet another embodiment of the present invention, the lubricatingfluid is used in the treatment of vaginal dryness and/or alsoinfertility related to vaginal dryness. The fluid, for example in gelform, would be topically applied to the vaginal mucosa. It should allowthe restoration of lubrication and consolidate mucus, thereby providingrelief and possibly facilitating the transport of male gametes as well.Thus, there is provided a vaginal lubricating composition comprising (orconsisting of) the above lubricating fluid. In embodiments, thecomposition also comprises one or more pharmaceutically acceptableadditive and/or therapeutic agent as defined above.

The present invention also provides the use of the above lubricatingfluid in lubricating a medical instrument. There is also provided amethod of lubricating a surface of medical instrument comprising thestep of contacting the lubricating fluid with said surface. Inembodiments, the medical instrument may be a syringe (for example thebarrel may be the surface to lubricate), an injection device, or anelution device. In such embodiments, the lubricating fluid preferablyconsists of pharmaceutically acceptable ingredients (i.e. polymers,solvents, additives, etc.) only.

According the results shown in the Examples below, after application,the fluid protects surfaces against wear without requiring any chemicalmodifications of the surfaces it protects. This is quite advantageous inthe case of biological tissues, as wear may cause pain. Currentlyexisting formulations of hyaluronic acid, such as injections ofhyaluronic acid (linear or crosslinked) have no demonstrated anti-weareffect. However, in the fluid of the invention, the combination of thebottle-brush polymer, which has a lubricating effect, with a linearpolymer that confers anti-wear protection results in a synergisticprotective effect that is greater than that which would be obtained bymerely summing the effects of the compounds taken separately.

In fact, the prior art fails to disclose lubricating fluids with bothstrong lubricating qualities and anti-wear effects.

Mixture of Polymers

There is also provided a mixture comprising:

-   -   a) a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b) a linear polymer,        wherein the bottle-brush polymer and the linear polymer are as        defined above.

In embodiments, the mixture is in solid form, for example in the form ofa powder.

The mixture may be used to produce the above lubricating fluid. Thismethod for producing the lubricating fluid comprises contacting themixture with a solvent and allowing dissolution of the mixture in thesolvent.

This method allows producing a required amount of the lubricating fluidaccording to demand for example at the location where it is used.

Optionally, the method further comprises mixing the mixture with thesolvent to speed the dissolution of the linear polymer and thebottle-brush polymer.

The lubricating fluid may be produced in vitro or in vivo.

In vitro production entails that the contacting, and optional mixingstep, carried out thereby before the lubricating fluid is used (e.g. forany of its above uses).

In vivo production entails, before said contacting step, theadministration of the mixture to a subject. Then, the dissolution occursin vivo. In such embodiments, the lubricating fluid preferably consistsof pharmaceutically acceptable ingredients (i.e. polymers, solvents,additives, etc.) only, i.e. the lubricating fluid is pharmaceuticallyacceptable. In preferred embodiments, the mixture and the liquid arecontacted at the desired site of action of the lubricating fluid. Thesolvent may be a body fluid or an extraneous solvent (i.e. a solventoriginating outside the subject, i.e. not a body fluid of the subject),such as those mentioned in the previous sections. In a particularembodiment, the mixture is administered orally in the form of an oralformulation that will release the linear polymer and the bottle-brushpolymer in the gastrointestinal tract. This allows the linear polymerand the bottle-brush polymer to contact and dissolve in a fluid of thegastrointestinal tract (which will thereby act as a solvent), therebyproducing the lubricating fluid in situ.

In embodiments, the mixture comprises the bottle-brush polymer and thelinear polymer present in a bottle-brush polymer:linear polymer weightratio between about 1:1 and about 1:20, preferably between about 1:2.5and about 1:15, more preferably between about 1:5 and about 1:10.

Surface Bearing a Polymeric Layer

There is also provided a surface bearing a polymeric layer comprising:

-   -   a) a acceptable bottle-brush polymer comprising a polymeric        backbone with polymeric pendant chains, and    -   b) a acceptable linear polymer,        wherein the bottle-brush polymer and the linear polymer are as        defined above.

In embodiments, the bottle-brush polymer and the linear polymer arepharmaceutically acceptable.

The surface may be for example a glass surface, a plastic surface, ametal surface, etc.

Examples of surfaces that can bear the polymeric layer include a surfaceof an ophthalmic lens, for example a contact lens, including either orboth sides of the lens. Other examples include a surface of an implant,such a joint replacement implant, including hip and knee replacementimplants. Yet other examples of surface include a the barrel of asyringe, a surface of an injection device, and a surface of an elutiondevice.

The polymeric layer can be manufactured on the surface by methods wellknown to the skilled person.

In embodiments, the polymeric layer can release the bottle-brush polymerand linear polymer, for example at a desired site of action, when incontact with a liquid, for example a biological liquid, so as to have alubricating effect. In such embodiments, the polymeric layer ismanufactured so that the bottle-brush polymer and linear polymer candetach from the surface at a desired rate. As an example, a solution ofthe polymers (e.g. for example the above lubricating fluid) could besolvent-casted on the surface.

In other embodiments, the bottle-brush polymer and linear polymer aremeant to remain on the surface for a period of time when in contact witha liquid, for example a biological fluid. Since the surface bear boththe bottle-brush polymer and linear polymer, it can impart a desiredlubricating effect, for example, at a desired site of action. In suchembodiments, the polymeric layer is manufactured so the polymers willnot detach from the surface or will become detached at a slow rate(including a very slow rate due to unavoidable wear and tear). As anexample, the polymers could be chemically grafted on the surface.

In embodiments, the polymeric layer comprises the bottle-brush polymerand the linear polymer present in a bottle-brush polymer:linear polymerweight ratio between about 1:1 and about 1:20, preferably between about1:2.5 and about 1:15, more preferably between about 1:5 and about 1:10.

Porous Material with Embedded Polymers

There is also provided a porous material having embedded therein:

-   -   a) a bottle-brush polymer comprising a polymeric backbone with        polymeric pendant chains, and    -   b) a linear polymer,        wherein the bottle-brush polymer and the linear polymer are as        defined above.

The porous material may be any porous material. Examples of porousmaterials include:

-   -   gels, including crosslinked gels (An example of a gel is a        hydrogel. An example of a hydrogel porous material is a contact        lens);    -   sponges; and    -   textiles and textile fibers, including woven and non-woven.        Examples of textiles and textile fibers those used in diapers,        facial tissues and dental floss.

Herein “embedded” means that the bottle-brush polymer and linear polymerare contained within the pores and crevices of the porous material.

When the bottle-brush polymer and linear polymer are embedded in aporous material, they are released when the material contacts is used incontact with a solvent for the polymer. Indeed, in use, the material canbe subjected to mechanical constraint, which will release the polymersand allow them to provide the desired lubricating effect.

In embodiments, the bottle-brush polymer, the linear polymer, and theporous material are pharmaceutically acceptable.

In embodiments, the porous material comprises the bottle-brush polymerand the linear polymer present in a bottle-brush polymer:linear polymerweight ratio between about 1:1 and about 1:20, preferably between about1:2.5 and about 1:15, more preferably between about 1:5 and about 1:10.

Definitions

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All subsets of values within the ranges arealso incorporated into the specification as if they were individuallyrecited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed.

No language in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Herein, the term “about” has its ordinary meaning. In embodiments, itmay mean plus or minus 10% or plus or minus 5% of the numerical valuequalified.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Herein, the term “pharmaceutically acceptable salt” refers to salts thatare pharmacologically acceptable and substantially non-toxic to thesubject to which they are administered. More specifically, these saltsretain the biological effectiveness and properties of the compounds ofthe invention and are formed from suitable non-toxic organic orinorganic acids or bases. The salts of the invention include base saltsformed with an inorganic or organic base. Such salts include alkalimetal salts such as sodium, lithium, and potassium salts; alkaline earthmetal salts such as calcium and magnesium salts; metal salts such asaluminium salts, iron salts, zinc salts, copper salts, nickel salts anda cobalt salts; inorganic amine salts such as ammonium or substitutedammonium salts, such as e.g. trimethylammonium salts; and salts withorganic bases (for example, organic amines) such as chloroprocainesalts, dibenzylamine salts, dicyclohexylamine salts, dicyclohexylamines,diethanolamine salts, ethylamine salts (including diethylamine salts andtriethylamine salts), ethylenediamine salts, glucosamine salts,guanidine salts, methylamine salts (including dimethylamine salts andtrimethylamine salts), morpholine salts, morpholine salts,N,N′-dibenzylethylenediamine salts, N-benzyl-phenethylamine salts,N-methylglucamine salts, phenylglycine alkyl ester salts, piperazinesalts, piperidine salts, procaine salts, t-butyl amines salts,tetramethylanmonium salts, t-octylamine salts,tris-(2-hydroxyethyl)amine salts, and tris(hydroxymethyl)aminomethanesalts.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1

What follows is a brief description of the procedure used to manufactureand test an embodiment of the present invention. In the followingprocedure, the term “polymer solution” refers to a solution containingthe bottle-brush polymer.

In this example, the design of lubricating and wear protecting fluidsbased on mixtures of bottle-brushes (BB) and linear polymer solutionsare described. FIG. 38 depicts the chemical structures of an exemplarybottle-brush polymer and sodium hyaluronate. To illustrate this concept,hyaluronic acid (HA), a naturally occurring linear polyelectrolyte, anda water-soluble synthetic BB polymer were used. Individually, these twopolymers exhibit poor wear protecting capabilities compared to that ofsaline solutions. Mixture of the two polymers in pure water or in salineallows the wear protection of surfaces over a wide range of shearingconditions to drastically increase. We demonstrate that this synergybetween the BB and HA polymers emerges from a strong cohesion betweenthe two components forming the boundary film due to entanglementsbetween both polymers. The example also show that this concept can beapplied to other types of linear polymers and surfaces and isindependent of the chemical and mechanical properties of the surfaces.

Here, we show that it is possible to design lubricating fluids able toprovide excellent wear protection without any chemical modification ofthe surfaces. The fluids use two components, BB polymers containingzwitterionic pendant chains synthesized by atom transfer radicalpolymerization, ATRP, and a natural linear polymer, sodium hyaluronate(HA). Both components are soluble in pure water or saline conditions.

A surface forces apparatus (SFA) was used to characterize the wearprotection capacity and the lubricating properties of the various fluidstested. The SFA allows measuring frictional forces under a wide range ofpressure and sliding speeds while monitoring the separation distancebetween the surfaces at ±0.5 Å resolution as well as the shape of thecontact. Muscovite mica is the substrate of choice in SFA experimentsmostly because of its optical transparency and atomic flatness. Herein,mica was also used due to its extreme propensity to suffer damage undermoderate shear conditions in water and saline conditions.

Materials and Methods

Materials

Methyl methacrylate (MMA, purity=99%, Sigma-Aldrich, USA) and2-(trimethylsilyloxy)ethyl methacrylate (HEMA-TMS, purity >9%,Scientific Polymer Products Inc., USA) were passed through a columnfilled with basic alumina prior to use. 2-Methacryloyloxyethylphosphorylcholine (MPC, purity ≥97%, Sigma-Aldrich, USA) wasrecrystallized from acetonitrile and dried under vacuum overnight atroom temperature before polymerization. Tetrahydrofuran (THF) was usedafter it was purified by tapping off from a solvent purification columnright. Ethyl α-bromoisobutyrate (EiBr, purity ≥98%, Sigma-Aldrich, USA),copper(I) chloride (CuICl, purity ≥99.995% trace metals basis,Sigma-Aldrich, USA), copper(II) chloride (CuIICl2, purity ≥99.995% tracemetals basis, anhydrous, Sigma-Aldrich, USA), 2,2′-bipyridyl (bpy,purity ≥99%, Sigma-Aldrich, USA), 4,4′-Dinonyl-2,2′-dipyridyl (dNbpy,purity ≥97%, Sigma-Aldrich, USA), potassium fluoride (KF, purity ≥99%,spray-dried, Sigma-Aldrich, USA), tetrabutylammonium fluoride (TBAF, IMsolution in THF, Sigma-Aldrich, USA) and α-bromoisobutyryl bromide(purity=98%, Sigma-Aldrich, USA were used without any additionalpurification. Solvents were purchased from Aldrich and used as receivedwithout further purification.

Ruby mica-sheets were purchased from S&J Trading Inc. (Glen Oaks, N.Y.,USA). Milli-Q quality water was obtained from a Millipore Gradient A10S10 purification system (resistance=18.2 MΩ·cm, TOC≤4 ppb). Phosphatebuffer saline (10 mM Phosphate, 150 mM NaCl and pH 7.4) was prepared inthe inventors' laboratory. Hyaluronic acids of different molecularweights were obtained from lifecore biomedical (Minneapolis, USA).

Equipment and Analysis

Proton nuclear magnetic resonance (¹H NMR) spectroscopy was performedusing Bruker 300 MHz spectrometer. In all cases deuterated chloroform(CDCl₃) was used as a solvent, except for the bottle-brush polymer whichwas analyzed using deuterated methanol (CD₃OD). ¹H chemical shifts arereported in parts per million (ppm) downfield from tetramethylsilane(TMS).

Apparent molecular weight and molecular weight distribution measurementsof polymers, except those of the bottle-brush polymer, were measured bysize exclusion chromatography (SEC) using Polymer Standards Services(PSS) columns (guard, 105, 103, and 102 Å), with THF or DMF as an eluentat 35° C. at a constant flow rate of 1.00 mL/min, and a differentialrefractive index (RI) detector (Waters). The apparent number-averagemolecular weights (Mn) and molecular weight distribution (Mw/Mn) weredetermined with a calibration based on linear poly(methyl methacrylate)(PMMA) standards and diphenyl ether as an internal standard. Absolutemolecular weights were determined using ASTRA software from WyattTechnology by GPC-MALLS containing RI detector (Wyatt Technology,Optilab rEX), viscometer detector (Wyatt Technology, ViscoStar), and amulti-angle laser light scattering (MALLS) detector (Wyatt Technology,DAWN EOS) with the light wavelength at 690 nm.

Hyaluronic acid apparent molecular weight and dispersity were assessedby aqueous SEC in 10 mM PBS, pH 7.4, 150 mM NaCl buffer using TSKgelcolumns (TSKgel G6000PW, particle size 12 μm, and TSKgel G2500PW,particle size 12 μm, Tosoh Biosciences LLC) at a constant flow rate of0.5 mL/min, and Multi-Angle static Light Scattering (DAWN HELEOS,Wyatt), and Refractometer (Optilab T-rEX, Wyatt). The absolutenumber-averaged molecular weights (M_(n)) and molecular weightdispersity (M/M) were determined with a dn/dc set at 0.16 mL/mg. Theresults are shown in the following table.

M_(w) R_(g) [nm]^(a) [g/mol] Dispersity 10 mM^(b) 150 mM 10 kDa HA 1.16× 10⁴ 1.26 — 16 ± 2 60 kDa HA 5.97 × 10⁴ 1.43 — 33 ± 1 300 kDa HA 3.25 ×10⁵ 1.54 71 69 ± 4 1.5 MDa HA 1.32 × 10⁶ 1.44 220 154 ± 12 ^(a)Measuredby static light scattering ^(b)from ref.^(1,2)

Absolute molecular weights were determined using ASTRA software fromWyatt Technology by GPC-MALLS containing RI detector (Wyatt Technology,Optilab rEX), viscometer detector (Wyatt Technology, ViscoStar),quasi-elastic-light-scattering detector (Wyatt Technology, QELS+) and amulti-angle laser light scattering (MALLS) detector (Wyatt Technology,HELEOS) with the light wavelength at 690 nm.

AFM imaging was performed in air using a MFP3D microscope from AzylumResearch (Santa Barbara USA). Standard silicon nitride tips were used toimage the polymer deposited on a mica substrate from an aqueoussolution. After drying, the polymer film was introduced in themicroscope and imaging was performed at a scanning speed of 1 Hz with atypical image size of 5×5 microns.

Bottle-Brush Polymer (PMPC B2) Synthesis

A molecular bottlebrush with a grafting ratio of about 50% ofhydrophilic (phosphorylcholine-, PMPC B2) grafts was prepared via‘grafting from’ approach.

The backbone for the brush was synthesized through equimolarcopolymerization of HEMA-TMS and MMA, resulting in the polymer(HEMA-MS)₆₀₀-stat-MMA₆₀₀ (B2) with DP˜1200. GPC characterization of B2showed the signal with M=132,000 and low dispersity: M_(w)/M_(n)=1.16(FIG. 2, solid line).

The subsequent functionalization of B2 with atom transfer radicalpolymerization (ATRPP) initiating functionalities yielded themacroinitiator (B2 MI) with M_(n)=163,000 and low dispersity:M_(w)/M_(n)=0.5 (FIG. 2, dashed line). ¹H NMR analysis of B2 MI was usedto determine the ratio of MMA and HEMA (functionalized or not)incorporated into the polymer (FIG. 3), proving incorporation of 50 mol% of HEMA (functionalized with TMS or not) into the backbone. Inaddition, the spectra showed incomplete functionalization of HEMAresulting in 40 mol % of ATRP initiator sites in B2 MI.

B2 MI was later used to graft hydrophilic pendant chains via ATRP, asshown in FIG. 4. The grafting of hydrophilic PMPC pendant chains (PMPCB2) was performed in methanol/acetonitrile mixture (70/30, v.v. %) at45° C., obtaining a PMPC B2 brush with the composition of(PBiBEM₅₄₀-g-PMPC₂₈)-stat-PHEMA₆₀-stat-PMMA₆₀₀.

The three-step synthesis scheme is shown in FIG. 4.

Synthesis of P(HEMA-TMS)₆₀₀-stat-MMA₆₀₀ (B2)

First, the backbone for the brush, P(HEMA-TMS)₆₀₀-stat-MMA₆₀₀, which isa statistic copolymer of methyl methacrylate and2-(trimethylsiyloxy)ethyl methacrylate, was synthesized as follows.

A dry 25 mL Schlenk flask was charged with ethyl α-bromoisobutyrate(EbiB) (5.8 mg, 4.4 μL 0.030 mmol), Cu^(II)Cl₂ (3.1 mg, 0.023 mmol),dNbpy (0.113 g, 0.276 mmol), HEMA-TMS (9.28 g, 10.0 mL, 45.9 mmol), MMA(4.59 g, 4.9 mL, 45.9 mmol) and anisole (3.2 mL). The solution wasdegassed by three freeze-pump-thaw cycles. During the final cycle, theflask was filled with nitrogen and Cu^(I)Cl (11.4 mg, 0.115 mmol) wasquickly added to the frozen reaction mixture. The flask was sealed,evacuated and back-filled with nitrogen five times, and then immersed inan oil bath at 70° C. Reaction was stopped after 67 h via exposure toair, reaching the degree of polymerization 1200 for the final polymer.The monomer consumption was calculated by the integration of the MMA andHEMA-TMS vinyl groups' signal (CHH═C—CH₃, 6.11 ppm or 5.56 ppm) againstthe internal standard (anisole, op-Ar—H, 6.91 ppm). The product waspurified by three precipitations from hexanes, dried under vacuum for 16h at room temperature, and analyzed by ¹H NMR spectroscopy. The ratio ofPMMA (s, broad, CO—O—CH₃, 3.54-3.68 ppm) to P(HEMA-TMS) (s, broad,OCO—CH₂, 3.90-4.17 ppm) peaks resulted in the polymer composition:P(HEMA-TMS)₆₀₀-co-PMMA₆₀₀. Apparent molecular weights were determinedusing THF GPC: M_(n)=132,000 and M_(w)/M_(n)=1.16 (solid line, FIG. 2).

Synthesis of PBiBEM₅₄₀-stat-PHEMA₆₀-stat-PMMA₆₀₀ (B2 MI)

In the second step, the HEMA-TMS repeat units of brush backbone (B2)were functionalized. The result was a statistic copolymer of methylmethacrylate and 2-bromoisobutyryloxyethyl methacrylate with only somenon-functionalized hydroxyethyl methacrylate (HEMA) repeat unitsremaining: PRiBEM₅₄₀-stat-PHEMA₆₀-stat-PMMA₆₀₀.

The polymer, B2, (3.00 g, 0.017 mmol of polymer; 9.90 mmol of HEMA-TMSunits), potassium fluoride (0.701 g, 11.9 mmol) and2,6-di-tert-butylphenol (0.204 g, 0.99 mmol) were placed in a 100 mlround bottom flask. The flask was sealed, flushed with nitrogen, andthen dry THF (30 mL) was added. The mixture was cooled in an ice bath to0° C., tetrabutylammonium fluoride solution in THF (IM, 0.05 mL, 0.05mmol) was injected into the flask, followed by the drop-wise addition of2-bromoisobutyryl bromide (2.73 g, 1.50 mL, 11.9 mmol). After theaddition the reaction mixture was allowed to reach room temperature andstirring was continued for 16 h. Next, triethylamine (1.0 mL) andanother portion of α-bromoisobutyryl bromide (0.4 mL) were added, andthe mixture was stirred for another hour. The solids were filtered off,and the solution was precipitated into methanol:water (70:30, v/v %).The precipitate was re-dissolved in chloroform and passed through ashort column filled with basic alumina. The filtrate was re-precipitatedthree times from chloroform into hexanes and dried under vacuumovernight at room temperature.

Apparent molecular weights were determined using THF SEC: M=163,000 andM_(w)/M_(n)=1.15 (dashed line, FIG. 2).

The NMR spectrum of this polymer is shown in FIG. 3.

Synthesis of (PBiBEM₅₄₀-g-PMPC₂₈)-stat-PHEMA₆₀-stat-PMMA₆₀₀ (“PMPC B2”or “B2 PMPC”)

As a last step, 2-methacryloyloxyethyl phosphorylcholine was polymerizedto form pendent chains grafted onto the functionalized repeat units ofthe backbone of the brush.

A dry 10 mL Schlenk flask was charged with polymer macroinitiator (B2MI) (0.0059, 0.0124 mmol of BiBEM groups), 2-methacryloyloxyethylphosphorylcholine (MPC) (1.10 g, 3.73 mmol), bpy (0.0066 g, 0.0422mmol), Cu^(II)Cl₂ (0.33 mg, 2.5 μmol), and acetonitrile/methanol (1.0mL/2.5 mL). The solution was degassed by three freeze-pump-thaw cycles.The flask was sealed, evacuated and back-filled with nitrogen and thenimmersed in an oil bath of 45° C. Then the degassed Cu^(I)Cl solution inmethanol (18.4 mg, 18.6 μmol in 1.0 mL methanol) was added to thereaction mixture. The polymerization was stopped after 1 h 15 min. byexposing the solution to air, achieving the brush with DP˜28 of PMPCpendant chains as determined by ¹H NMR. The brush was purified bydialysis against methanol using a 25,000 MWCO membrane. The PMPC B2brush was obtained as white powder.

Formulation of Lubricating Pharmaceutical Fluids

10.0 mg of different molecular weight hyaluronic acid (HA) (1.5 Mda, 500kDa, 60 kDa and 10 kDa) were dissolved with magnetic stirring in 10 mLMilli-Q water or 10 mM PBS pH 7.4 in a glass vial. The solution was keptat 4° C. for 24 h prior to use. 1 mg/mL solution of PMPC B2 was preparedin the same buffers. 50 μL of the polymeric solution was added to 450 μLof HA solution resulting in a solution of PMPC B2 at 100 μg/mL and HA at0.9 mg/mL and was homogenized with a vortex for 1 min. The solution wascentrifuged at 14,000 rpm during 10 min to remove aggregates, particlesor dust. For each SFA analysis, 50 μL of corresponding pharmaceuticalfluid was injected between the surfaces. Surfaces were then let toequilibrate for 1 h prior to measurements.

Surface Forces Measurements

Normal Interaction Forces

Measurements of the normal interaction forces between two opposingsurfaces as a function of the separation distance were carried out usinga Surface Forces Apparatus (SFA 2000, SurForce LLC, USA). The normalinteraction force F^(⊥) is determined by measuring the deflection of thespring cantilever (spring constant of 482 N/m) supported by the lowersurface. The distance between the surfaces is measured using MultipleBeam Interferometry (MBI). Fringes of Equal Chromatic Order (FECO) aregenerated using white light multiple beam interferometry shining whitelight through two back-silvered mica sheets glued onto glass cylinders(radius of curvature ˜1.5 cm). FECO are analyzed in a spectrometerequipped with a CCD camera (Andor Zyla, Germany). The separationdistance D between the surfaces is calculated (to ±1 Å) from thewavelength of the interference fringes. The two disks were mounted inthe SFA chamber in cross cylinder geometry and brought into mica-micaadhesive contact in dry air in order to determine the referenceposition. Afterward, the cylindrical disks were separated by roughly 1mm and lubricating pharmaceutical fluid was injected between thesurfaces. Immediately after injection, the bottom of the SFA chamber wasfilled with water in order to saturate the surrounding vapors and tolimit evaporation of the injected liquid. The normal interaction forcesbetween the two polymer coated surfaces as a function of surfaceseparation were determined on approaching (compression) and separating(decompression) the surfaces. For each test, all force runs (in and out)were performed at least in triplicate with the motor or thepiezoelectric tube at a speed range of 0.4-1.6 nm/s. Each experiment wasreproduced 2 to 6 times.

The FAS setup used is shown in FIG. 5.

Friction Force Measurements

The friction force F_(II) was measured by moving the lower surfacehorizontally and measuring the response of the upper surface. Beforemeasuring the friction forces, three cycles of normalcompression/decompression were performed on the same contact position.For friction tests, a piezo bimorph drove the lower surface in a backand forth motion at a constant sliding frequency of 50 mHz controlled bya function generator. After sliding was finished, the load between thesurfaces was slightly increased. The friction force transmitted to theupper surface was detected by semi conductive strain gauges, amplifiedby amplifiers and digitally recorded. Acquired data were processed usingOrigin Lab® software. The normal force was measured using calibratedstrain gauges installed on the double cantilever spring supporting thelower surface. Separation distance and surface deformation werecontinuously recorded during the experiment using the FECO fringesanalysis as described in the previous section. The pressure is assessedby dividing the normal force right before polymeric layer break andsurface of contact measured by the flat area of contact fringes.

Speed Effect Measurement

The previously described setup is used to assess the speed effect.Surfaces are brought into contact and set at a constant load which isrecorded throughout the experiment by strain gauges. Using the functiongenerator, a piezo bimorph drove the lower surface in a back and forthmotion. After sliding at one frequency was finished, the frequency wasincreased. The frequencies that were used in the experiments were 0.5mHz, 1 mHz, 10 mHz and 50 mHz. The friction force was measured usingcalibrated strain gauges installed on the double cantilever springsupporting the lower surface.

Chitosan Gels and Tribotesting

A 2.5% w/w chitosan solution (Mw=6.04×10⁵, Mw/Mn=1.64, DA 4.3%) wasprepared by dissolving the polymer in an aqueous acetic acid solution.Air bubbles were removed by centrifugation, and the highly viscoussolution was compression-molded to obtain a slab of constant thickness.The chitosan solution was then placed in a 1 M NaOH coagulation bath tocomplete gelation. Gel disks of 11 and 21 mm in diameter were obtainedusing biopsy punchers and neutralized in pure water until use.

For the tribotesting experiments, the 11 mm diameter gel disk was gluedon the top mobile part of a custom-made tribometer. The larger gel diskwas glued on a metallic immobile bath filled with the tested polymersolution and left to incubate for 1 h prior to experimentation. Normaland tangential forces were recorded and analyzed with a homemade routineprogrammed in Labview. Roughness of the gels was quantified afterperforming tribotesting using an interferometric microscope.

Results

Characterisation of the Bottle Brush Polymer

FIGS. 6 A) and B) shows AFM pictures of the bottle-brush polymer on micain air. The AFM pictures show the bottle-brush polymer's worm-likestructure, as expected for a bottle-brush polymer. FIG. 6 also showsthat the polymer chains accommodate themselves to form a homogeneousfilm on the mica surface.

The contour length of the bottle-brush polymer is shown in FIG. 7.

In addition, the molecular weight of the backbone of the bottle-brushpolymer was 88700 Da, while the molecular weight of the pendant chainwas about 13275 Da.

Load Bearing Capacity of the Fluid

Load bearing capacity is the ability of a fluid film to sustain a normalstress without breaking. It can be assessed either by measuring thenormal pressure required to bring two surfaces to atomic contact or bymeasuring the separation distance between the two surfaces at a givenapplied pressure. This property is critical to evaluating the ability ofthe fluid to protect the surfaces against impact damage and wear.

FIG. 8 shows the interaction forces measured in the SFA between two micasurfaces across synovial fluid (SF, bovine), HA solution (1 mg/mL),bottle-brush polymer solution (1 mg/mL) and a HA/bottle-brush polymersolution at a 1:1 ratio. The load bearing capacity of the confined filmwas assessed by measuring the separation distance between the surfacesat a normal force of 20 mN/m (corresponding to a normal pressure ofapproximately 1 Mpa). The results show that HA alone has a very poorload bearing capacity compared to SF (d_(HA)<<d_(SP)). The bottle-brushpolymer (noted as “polymer” in FIG. 8) exhibits a stronger load capacitythan HA but fails to reach SF. The bottle-brush polymer: HA mixturedemonstrates the highest load bearing capacity, far superior to the sumof the two components alone(d_(bottle-brush polymer:HA)>>d_(bottle-brush polymer)+d_(HA))confirming that a synergistic interaction between HA and the polymerallows the confined film to sustain much more pressure than SF itself.

We measured the normal interaction forces, F, between two facing micasurfaces of curvature, R, in the presence of the different components ofthe lubricating fluids, first individually and then mixed together. Tocover a wide range of conditions, we tested different HA molecularweights, M, in pure water and in phosphate buffer saline (PBS, 150 mMfor low ionic strength and 1500 mM NaCl for high ionic strength, both atpH 7.4). The interaction forces were recorded as a function of theseparation distance, D, between the surfaces starting from severalhundred nanometers (zero interaction regime) down to a few angstroms(strong interaction regime) in order to capture the full interactionforce profile (force law) of the system.

Interaction forces measured in the SFA were obtained in presence of HAsolutions in pure water and in salines. Each interaction force profilewas measured at least three times on the same contact point and repeatedover multiple contact points. Prior to the first measurement, andequilibration time of one hour was set for all tested conditions.

FIGS. 9 and 10 shown the force profiles measured in HA solutions in purewater and in saline buffers. HA being negatively charged under alltested conditions; it is expected to adsorb as a random coil onnegatively charged mica surfaces through the formation of hydrogenbonds.

As shown in FIG. 9, force profiles present two distinctive trends,depending on the ionic strength of the medium. In pure water, the onsetof the interaction forces was located between 20 and 30 nm independentlyof the molecular weight of HA (see FIG. 10). Such weak dependence on themolecular weight suggests that, during the time window of theexperiment, only low molecular weight chains could adsorb on thesurfaces (a case similar to the Vroman effect). Low molecular weightmolecules are expected to adsorb first on mica surfaces because they aremore mobile. Later, larger molecules, which have higher affinity for thesurface, are expected to displace them. In saline, the interactionforces exhibited shorter range forces starting between 5 and 10 nm.These short-range interaction forces systematically presented periodicinstabilities, indicating the presence of a layered structure at thesurfaces (see inset of FIG. 9). The characteristic size ΔD of theseinstabilities was ΔD=0.2-0.3 nm, in agreement with the size of a watermolecule. Adhesive forces were systematically measured upon separationof the surfaces independently of the medium (pure water or saline).

These observations demonstrate that, under the present experimentalconditions, HA does not strongly bind to the mica surfaces in saline dueto the presence of a 2-3 nm thick hydration layer strongly interactingwith the surface, while in pure water, the polymer can adsorb stronglyand form a stable soft layer.

Interaction forces in the presence of the BB polymer alone werestrikingly different from those of the HA polymer alone (see FIG. 11).Repulsive forces were measured on approach and separation of thesurfaces, independently of the ionic strength of the medium. Moreinterestingly, the force profiles were insensitive to the ionic strengthof the medium. Interestingly, the conformation of linear polymer chainsof MPC has been reported to be insensitive to the ionic strength insolution²⁰ and at surfaces.²¹ The onset of the interaction forces(determined at F/R=0.01 mN/n) was found to vary between 100 and 125 nm,independently of the medium ionic strength (FIG. 11). Given that thecontour length of the polymer, assessed by AFM imaging in air (see FIG.7), is ˜140 nm, a significant part of the BB polymer is expected to beextending toward the medium.

Interaction forces under high confinement (D<10 nm) did not present anylayering transition or any evidence of hydration forces, which confirmsthat the BB polymer interacts strongly with the hydrated surface layer,strongly enough to displace the water molecules present at the surface.Such observations echo some reports showing that charged amine headgroups adjacent to H-bonding donor groups can efficiently remove boundwater from a hydrated surface and facilitate H-bonding. No adhesiveforces were measured upon separation of the surfaces.

Force profiles of the different polymer mixtures were also measured.Each HA:BB polymer mixture was tested in three different media asdescribed in the manuscript. Force profiles were recorded on threedifferent contact points to ensure good reproducibility.

Interaction forces across HA-BB polymer mixtures in saline presentedfeatures similar to those of BB polymer alone (FIGS. 12 and 13). Anexception was the mixture containing HA 10 kDa; the onset of theinteraction forces measured for the different polymer mixtures rangedbetween 120 and 180 nm depending on the medium, which is similar to theonset measured with the BB polymer alone and at least twice the valuemeasured for any of the tested HA alone solutions. Below a separationdistance D≈50 nm, a steep increase in the interaction forces wassystematically observed, suggesting the presence of a dense/stiff layerof polymer at the surfaces. The thickness of this dense (proximal)polymer layer was found to be highly sensitive to the ionic strength ofthe medium. Based on the force profiles, the thickness of the proximallayer can vary from approximately 20 nm at 150 mM NaCl to 5 nm at 1500mM NaCl. No adhesive forces were measured if the surfaces were separatedat D>50 nm (in the distal region of the interaction profile), and weakadhesive forces were systematically observed when separating thesurfaces at D<50 nm. These observations confirmed that the proximallayer contains mainly HA.

These force profiles demonstrate that the polymer mixtures form aninterpenetrated layered thin film as represented in FIG. 14. Theproximal layer of such film contains most of the HA molecules adsorbedat the surface and portions of BB polymer chains, whereas the distallayer is composed solely of BB polymer molecules extending in themedium.

Similarly, FIG. 15 shows a simplified modelized structure of thedeposited polymer film as observed in the SFA. A thin film of HA iscovering the mica surface together with the bottle-brush polymer. Thelatter extends in the medium, giving rise to the observed long-rangeinteraction forces measured in the SFA. The short-range part of theinteraction forces is dominated by the compression of HA and thebottle-brush polymer.

In pure water, the force profiles of the different mixtures did notexhibit any marked transition between the HA-rich proximal layer and theBB polymer distal layer. Instead, the force profiles show a continuousincrease, consistent with an extended proximal layer fully overlappingwith the distal layer.

Tribological Properties

After measuring the normal interaction forces in the different media, wecharacterized the tribological properties of the different polymermixtures. In a first series of experiments, we measured the frictionforce, F_(S), as a function of the applied normal force, F_(N) (FIGS. 16and 17). For all the tested conditions, F_(S) was found to increaselinearly with F_(N) until damage of the surfaces occurred (see FIG. 16).We therefore defined the friction coefficient of our system asμ=F_(S)/F_(N). In saline only (no polymer added), frictional forces werevery weak (not shown), giving a friction coefficient of μ=0.002±0.001(FIG. 17), in good agreement with previous reports.²⁴ Using opticalinterferometry,²⁵ we measured the critical pressure, P*, at which theonset of surface damage was triggered. Onset of damage appeared assudden crack formation and propagation along the direction of shearing.We found a value of P*=0.73±0.03 Mpa for both saline conditions. We alsofound that p increased 2 orders of magnitude after damage occurred andranged between 0.2 and 0.7, as shown in FIG. 17. On the other hand, inpure water, damage of the surfaces occurred almost immediately after afew shearing cycles, indicating that P*≈0 Mpa. These results echo recentstudies demonstrating that, in saline medium, surface-adsorbed ionsfacilitate the formation of a lubricating water layer able to sustain asignificant amount of normal pressure under shear and thereforeprotecting the surfaces from damage.^(26,27)

In the presence of HA, the measured values of μ in saline before damage(P<P*) were found to be independent of the molecular weight of thepolymer (FIG. 17) and close to the values found in saline only (μ≈10⁻³).

To elucidate if polymer chains were still present in between thesurfaces at the onset of wear, we monitored the film thickness and therefractive index of the confined film during shear (FIG. 18). As theapplied pressure increased (not shown in the figure) the film thicknessdecreased and concomitantly, the refractive index of the confined filmincreases slightly from 1.33 (bulk water) up to ˜1.47. Experimentsperformed in PBS and in buffered HA present the same trend suggestingthat HA is quickly depleted from the contact as the normal pressure isapplied. In summary, measurements of the thickness and refractive indexof the confined film before damage show that the contact area is quicklydepleted of polymer, leaving only adsorbed ions and water molecules atthe interface (see FIG. 18).

The value of P* for HA was ≈0.7 Mpa, independently of its molecularweight, which is identical to the value encountered in saline only andconsistent with the previous observation of HA being depleted from thecontact before damage occurs. In pure water, HA solutions demonstratedvery poor stability and systematically led to the formation of polymeraggregates in the shearing contact. These polymer aggregates lead tofocal pressure increase throughout the contact area and eventuallytriggered crack formation. As a consequence, the measured value of P*was ≈0 Mpa even though μ=0.02 for all of the M_(w) tested after damageoccurred.

Frictional properties of the BB polymer alone in pure water and salinewere drastically different from those of HA or saline alone (FIG. 17).The measured friction coefficient μ before damage was 1 order ofmagnitude higher than that of HA or saline alone and was equal toμ=0.03±0.01, independently of the ionic strength of the medium. Thisresult is consistent with the above observation from the normal forceprofiles showing that BB polymer adsorption and conformation on micawere independent of the ionic strength. In saline and pure water, themeasured values of P* were 0.25±0.02 and 0.56±0.04, respectively, whichis lower than that of HA and saline alone under similar conditions.

As shown in FIGS. 16 and 17, mixing HA and the BB polymer did notimprove significantly the lubricating properties of the surfaces. Themeasured friction coefficient of the different mixtures before damage(P<P*) was independent of HA molecular weight and equal to μ=0.02±0.01,independently of the medium's ionic strength. This observation suggeststhat the friction coefficient of the mixture is solely controlled by thepresence of the BB polymer when P<P*.

FIG. 17 shows the values of friction coefficients obtained with HA aloneand with the bottle-brush polymer. Addition of the bottle-brush polymerincreases the friction coefficient from 2.10⁻³ to 2.10⁻², similar to thefriction coefficient of the bottle brush alone. As shown later, theincrease in friction coefficient for the mixture is inversely correlatedto the wear protection imparted by the fluid to the surfaces.

FIG. 19 shows the evolution of the critical pressure at which surfacedamage occurs under different conditions. In pure water, HA alone doesnot sustain any significant normal pressure. Addition of the bottlebrush polymer increases the wear protection dramatically, especially athigh molecular weights of HA. This same effect was observed in salineconditions, albeit to a smaller magnitude due to the protective effectof the hydration layers on the surfaces generated by adsorbed surfaceions. Indeed, the value of P* for each of the components alone was belowthe value of pure PBS independently of HA molecular weight, while themixtures presented significantly higher value of P*, especially at highHA M_(w).

Most interestingly, the value of P*, which relates to the wearprotection capacity of the polymer mixture, was highly sensitive to HAmolecular weight. As can be seen in FIG. 19, for mixtures with a finalconcentration of 100 μg/mL of BB polymer and 1 mg/mL of HA (1:10 massratio), P* increased significantly with HA molecular weight asP*∝log(Mw). In pure water, the HA-BB polymer mixture led systematicallyto a significant increase in wear protection, especially at high HAmolecular weight, with P* increasing from 0 Mpa in the absence of BBpolymer to 3.2 Mpa in the presence of BB polymer. A similar trend wasobserved in saline solutions, with a 2-fold increase of P* at thehighest HA molecular weight in the presence of the BB polymer.

To obtain more insights into the mechanism underlying such a phenomenon,we monitored the evolution with shearing time of the film thicknessunder different shearing conditions. Measurements of the thin filmthickness during shear in pure water and in PBS shown in FIGS. 20 and 21show that the polymer mixtures are able to sustain significantly morepressure compared to the polymers alone and in a Mw-dependent manner.

More specifically, FIG. 20 shows that, as the normal pressure P isincreased, the film thickness, D, increases dramatically when the mediumcontains HA only in pure water, indicating the immediate aggregation ofthe polymer and the triggering of surface wear. In saline (FIGS. 21 and18), the data show that HA is quickly depleted from the contact, leadingto a rapid decrease of D down to 0.5 nm before damage occurs at P═P*.

In the presence of BB polymers alone, the film thickness at P═P* was 1nm for both saline conditions, which is thicker than the previouslymentioned value obtained for HA solutions. Such a high value of the filmthickness indicates that BB polymer chains are still present in thecontact at the onset of wear. Similar observations were confirmed withthe different polymer mixtures, although the values of P* weresignificantly higher than BB or HA alone (FIGS. 20 and 21). Thesignificant increase of P* in the case of the polymer mixturescorrelates with the higher film thickness at the onset of damage, whichindicates the existence of strong intermolecular interactions between HAand the BB polymer. Such interactions maintain a strong cohesion betweenthe different polymer chains under shearing conditions and allows theconfined film to sustain significantly more normal pressure.

To elucidate the nature of the interactions responsible for such strongintermolecular cohesion, we performed a series of isothermal titrationcalorimetry experiments (ITC). Isothermal titration calorimetry of theBB polymer in different polymer solutions was performed using a VP ITCfrom MicroCal. Running on Origin® 7. In the syringe, a buffered BBpolymer solution was loaded at a concentration of 0.6 mg/mL and in thereceptor cell, a buffered solution of HA or PVP at 1 mg/mL was loaded.All solutions have an ionic strength of 150 mM and were degassed prioruse. Experiments consisted in 25 injections of 10 uL each in thereceptor cell (1.42 mL) at an injection speed of 2 uL/s and agitationspeed of 300 rpm. As a control, the BB polymer solution was alsotitrated in buffered saline to obtain the dilution heat of the polymer.The results, at FIG. 22, show no differences between the titrationexperiments in HA or PVP and buffered saline, which demonstrate thatthere is no direct interaction between the polymers.

Indeed, no thermal signature was measured during mixing of the polymers,indicating that no detectable interaction (electrostatic or hydrophobic)exists between the two polymers. Therefore, the important role played byHA molecular weight in tuning the cohesive strength of the filmdemonstrates that chain entanglements are the main factor responsiblefor the polymer film cohesion (FIG. 23).

In order to demonstrate the generality of the mechanism and its broadapplication, we performed a second series of tribological tests toestablish the impact of shearing speed, BB/HA polymer ratio, polymerchemical structure, and surface chemistry (see FIG. 24 to 28).

FIG. 24 shows that wear protection imparted by the BB polymer-HA mixturewas not affected by the sliding speed of the surfaces. At a pressure of0.5P*, no damage of the surfaces was observed even when varying thesliding speed by 3 orders of magnitude. Indeed, as shown in FIG. 24,varying the sliding speed over three decades, between 0.01 and 10 μm/sat P<P*, did not trigger any damage of the surfaces, indicating a weakdependence, if any, of P* on the sliding speed.

FIG. 25A) shows that BB/HA (1.5 MDa) ratio has a significant impact onP* and was found to be optimum at 1:10. Indeed, in FIG. 25A), we showthat P* depends strongly on the BB/HA ratio and is optimum at a ratio ofBB/HA=1:10 (mg/mg). Above this optimum ratio, the value of P* is equalto the value of HA F alone, indicating that HA has displaced the BBpolymer from the surface. Below the optimal ratio, the value of P* isequal to the value obtained for BB alone, indicating that BB polymer isthe sole component in the confined film.

FIG. 25B) shows that HA can be replaced by PVP to obtain similarsynergistic wear protection when mixed with the BB polymer. Indeed, asimilar synergistic behavior between BB polymer and HA was also observedwhen replacing HA with poly(N-vinylpyrrolidone) (PVP), a neutral,water-soluble polymer (M=40 kDa). FIG. 25B) shows that the value of P*in saline (150 mM, pH 7.4) exhibits a 2-fold increase with a 1:10mixture ratio of BB/PVP compared to PVP alone. Similarly to HA, PVP didnot show any direct interaction with the BB polymer by ITC (FIG. 22).

The HA-BB polymer mixture was tested against mica/gold tribo-pair, aswell—see FIG. 25C). The wear protection enhancement was similar to thatof mica/mica. Gold being a ductile metal, its tribological propertiesare very poor in terms of wear resistance. For the tribo-pair mica/gold,P* was inferior by 1 MPa in the presence of HA or BB polymer alone. Asshown in FIG. 25C, the polymer mixture was once again significantly moreefficient at protecting the surfaces compared to the single componentsalone.

In all the tested conditions, the value of P* associated with thepolymer mixture is systematically superior to the sum of the valueassociated with the polymers alone, indicating a true synergisticinteraction between both components in terms of wear protection.

We finally tested the lubricating fluids between macroscopic hydrogelplugs of chitosan as model soft polymeric surfaces (FIGS. 26 to 28).Chitosan hydrogels have been extensively tested as cellular scaffoldsfor tissue engineering applications, but their poor resistance againstabrasive wear has hampered their translation to clinical settings.Tribo-testing of the hydrogels (2.5 w %) has shown that the cumulateddissipated energy, Ed, which is directly related to the wear volume,W_(v), is strongly diminished in the presence of the HA-BB mixturecompared to each component alone. Concomitantly, the surfaces'roughness, S_(a), was found to significantly decrease in the presence ofthe mixture compared to all other conditions due to surface polishingand restructuring.

Further Results

FIG. 29 shows an example of FECO fringes with no damage (up) and damagedappearance (down). The shape of the FECO fringes is indicative of theshape of the surfaces' contact point. Slight deformations in thefringes' shape indicate the appearance of a third body trapped inbetween the surfaces. Said third body can be generated by theaggregation of the confined polymer film under shearing conditions, orit could be composed of mica particles formed by abrasive wear of thesurfaces. In any case, these changes in contact geometry can be used totrack the onset of any kind of wear process occurring during atribological measurement without the need of separating the surfaces orthe use of any extra imaging techniques.

Effect of HA/BB weight ratio, and testing of other linear polymers (inPBS, HA Mw=1.5 MDa, PVP Mw=35 kDa)

Mixture Pressure of composition rupture (MPa) PBS only 0.7 HA only 0.7HA:BB 1:1 0.4 HA:BB 10:1 1.6 HA:BB 100:1 0.7 PVP only 1 PVP:BB 10:1 1.9(HA = hyaluronic acid, BB = bottle-brush polymer as described above. PVP= poly(vinlpyrrolidone)

Example 2—In Vitro Stability

We also evaluated in vitro stability of the bottle-brush (B1) polymer.

The bottle-brush polymer used for Examples 2 and 3 was similar to thatof Example 1, except that it had a backbone comprising 370 units ofmethylmethacrylate (MMA) and 459 units of hydroxylethylmethacrylate(HEMA), with pendant chains containing 35 unit of 2-methacryloyloxyethylphosphorylcholine (MPC) grafted on the hydroxylethylmethacrylate repeatunits. The grafting ratio was 0.55. In other words, the BB polymer was(PBiBEM₄₅₆-g-PMPC₃₅)-stat-PHEMA₃-stat-PMMA₃₇₀.

The BB polymer (white powder) was dissolved at 100 μg/mL in a homemadePBS composed of 10 mM phosphate ions and 150 mM NaCl with a pH 7.4. Thepolymer was left this saline solution in a dark container at 4° C., 22°C. or 37° C.

A Surface Forces Apparatus (SFA 2000, SurForce LLC, USA) was used tomeasure the normal force profiles of the BB solution. Back-silvered micasheets were glued (epoxy glue Epon™ 1004F) on glass cylinders with acurvature radius, R, of 2 cm under a laminar flow hood. The cylinderswere mounted in SFA chamber in a cross-cylinder configuration. The SFAchamber was then purged with dry nitrogen and the surfaces were broughtinto adhesive contact to measure the zero contact using a springcantilever with a spring constant of 482 N/m.

The separation distance between the two opposing mica surfaces wasdetermined from the FECO fringes using mica-mica contact. The surfaceswere then separated and 50 μL of BB polymer solution were injectedbetween the surfaces and pure water was injected in the chamber tosaturate the vapors to prevent solution evaporation. The setup was leftto equilibrate for 1 h. The normal interaction forces, F_(N), wererecorded as a function of separation distance, D, for in (compression)and out (separation) runs at a speed of 0.002 μm/s. The fringes wereanalyzed using a Matlab software. Experiments were performed at leastthree times at different contact positions.

FIG. 30 shows the onset of interaction as a function of time atdifferent temperature. FIG. 31 shows the kinetic constant as a functionof 1/temperature. The results shown in these figures confirm that thepolymer conformation at the surface changes with time and temperaturemostly due to the loss of pendant chains from hydrolysis. Arrhenius plotshows that the activation energy of the degradation reaction isconsistent with a hydrolysis reaction.

Example 3—In Vivo Tests

Materials and Methods

Rat Model of Osteoarthritis Animals

Eight-week-old male Lewis rats were purchased from Charles River Canada(Saint-Constant, QC) and housed under standard conditions. They werehoused at 25° C. with a 12:12-hour light-dark cycle and provided with astandard laboratory diet and water ad libitum. The experimental protocoland all animal procedures were carried out in accordance with theguidelines of the Canadian Council on Animal Care (CCAC) and wasapproved was approved by the Institutional Animal Care Committee at theResearch Center of Sainte-Justine University Hospital, Montreal, Canada.

Study Design

The study was conducted as a fractional factorial experiment. Animalswere submitted to surgery anterior cruciate ligament transection (ACLT)was performed on the right posterior knees, and no surgery (negativecontrol) on the left posterior knees. Subsequently, animals wereassigned to one of two treatment groups, as detailed below (Table 1),with 2 subjects per group.

TABLE 1 Experimental groups Group Number Surgery Treatment 1 ACLT HA (2mg/ml) in PBS (50 microliters) 2 ACLT HA (2 mg/ml) in PBS (50microliters) 3 ACLT HA (2 mg/ml) + BB (350 μg/ml) in PBS (50microliters) 4 ACLT HA (2 mg/ml) + BB (350 μg/ml) in PBS (50microliters) HA: Hyaluronic acid BB: Bottle-brush polymer as describedin Example 2.Surgical Technique

OA was induced by surgical transection of the right anterior cruciateligament. The procedure was modified from previously published reports(Appleton et al Arthritis Research & Therapy 2008 10:407). We publishedthis surgery technique in Kaufman et al Arthritis Research & Therapy(2011) 13:R76. Animals were anaesthetized with inhaled isoflurane (3% 1L 02 induction in chamber, 2% 1 L 02 maintenance with face-mask), andprepared for surgery by clipping the hair over the ventral and medialaspects of the right leg from hindpaw to hip. The skin was disinfectedwith povidone-iodine, and a 3-cm incision was made medial to thepatellar tendon (FIG. 32A)). The subcutaneous tissue and muscle werethen incised and the patella laterally sublaxed; the joint capsule wasopened with the limb hyperextended (FIG. 32B)). With the limb in fullflexion, the anterior cruciate ligament was visualized by bluntdissection, and sectioned by a latero-medial eut parallel to the tibialplateau, using a \#11 scalpel blade (FIGS. 32C) and D)). Transection wasconfirmed with the anterior drawer test (FIGS. 32E) and F), E depictingthe knee before, and F) showing an anterior drawer). The patella wasthen replaced, and the limb extended (FIG. 32G)). The joint capsule(FIG. 32H)) and muscle layers (FIG. 32I)) were closed with 4-0polygalactin absorbable suture (horizontal mattress stitch, FIG. 32J)).50 μL of lidocaine was then injected into the joint capsule to providelocal analgesia. Skin was closed with steel surgical staples (FIG.32K)). Post-operative hydration (6 mUkg saline) and systemic analgesia(0.1 mg/kg buprenorphine HCl) were provided by subcutaneous injection.Surgical staples were removed 7 days post-operatively (FIG. 32L)).

Drug Treatment

Over the course of two weeks post-operatively, animals were treated byweekly intra-articular injections of 50 microliters of HA (2 mg/ml) inPBS, or HA (2 mg/ml)+BB (350 μg/ml) in PBS. The total volume was 50 μL.Injections were performed under isoflurane anaesthesia, using a 28 Gneedle. The compounds were injected into the right knee. All Injectionswere performed under isoflurane anaesthesia, using a 28 G needle.

FIG. 33 shows the intra-articular injection into the rat knee: Theshaved knee was maintained in extension (FIG. 33A))), the needle wasinserted under the patellar tendon into the joint space (FIG. 33B)), thesyringe plunger is depressed slowly (FIG. 33C)), successful injectionwas detected by an acute swelling of the articular space (FIG. 33D)).Photographs were taken with a 300-mm macro lens (approximatemagnification 2, as described in Kaufman et al. 2011, supra).

In Vivo Micro-CT Scanning

Micro-CT Scanning

Micro-computed tomography (micro-CT) is an efficient tool for the studyof bone morphometry and 2D/3D image analysis. In particular, itconstitutes a valuable tool for the non-destructive evaluation oflaboratory animals and the in vivo tracking of anatomical changes inbone, bone mass and bone microstructure. In this study, we analyzed thegeometrical parameters of the rat knee aiming to quantify the intraarticular space in control and experimental conditions.

We used a Skyscan 1176 micro-CT imaging (Skyscan, N.V., Belgium) scannerwith rotatable X-ray source and detector. After CO₂ asphyxiation,followed by decapitation, both left (control) and right (surgery) kneeswere removed, and scanned by the micro CT. To do so, right and leftknees were separately into a cylindrical Styrofoam holder placed in acarbon fiber half-tube bed of the Skyscan 1176. This was done in orderto position the limb at the scanning midline during scanning.

Image acquisition parameters were the followings: X-ray source voltage65 kV, current 384 μA, full X-ray power, and 1-mm thick aluminum filterfor beam hardening artifact reduction. The pixel size was 18 μm for a2,000×1,336 CCD detector array. The exposure time was 350 ms, therotation step 0.5°, with 1 frame averaging, and gantry direction in CC.The total scanning time was 15 min. During acquisition, the scanningconsisted of a stack of 720 images. The acquisition covered the regionof the knee joint from just above the proximal tibia and extended up tothe tibiofibular joint. Cross-section images were reconstructed using afiltered back-projection algorithm (software NRecon, v.1.6.10, Skyscan,Kontich, Belgium). For each scan, a stack of 1,328 cross sections wasreconstructed corresponding to a total reconstructed height of 10 mm,starting from the knee joint and extending distally along the tibialdiaphysis, with an interslice distance of 1 pixel (17.48 μm). Thereconstructed images were of 772×772 pixels each, 17.48 μm pixel size,and were stored as 8-bit images (256 gray levels).

Using the micro-CT, we first evaluated the impact of surgery (OAdevelopment) and then we quantified the spaces between the two bones:among 1200, 70-80 images were chosen where distances between the distalfemur and the proximal were less than 900 □m. Six images of each groupwere then analysed, performing 8-10 measurements on each image

To compare the values of the intra-articular space (space between thefemur and tibia) a paired Student's t-test was performed. Thiscomparison was performed to assess the possibility of the increasedspace effect of polymer HA, compared to the HA+BB. Results wereconsidered to be statistically significant for p<0.05.

Knees were then transported in a humidified chamber (with PBS solution)for the 3D mechanical topographical mapping analysis that was performedduring the next 24 h.

3D Mechanical Toporaphical Mapping

3D mechanical topographical mapping established cartilage thicknessvariation in rat joint. These analyses were performed in order todetermine if cartilage thickness was modified by intra articulartreatment with HA and HA+BB. We compared the control (left joints, tothe joints treated with HA. Cartilage thickness was investigated bytopographic variability of the mechanical properties of cartilage overthe articular surface (the thickness of the layer corresponding to thecartilage was measured with the XY Scan using a needle penetrating thesurface vertically until to the bone).

Mechanical Properties

Mapping of the mechanical properties of cartilage joints were performedfollowing already published procedures [Sotcheadt S. et al. J.Orthopaedic Res. 2016 DOI: 10.1002/jor.23330] Briefly, mechanicalproperties were measured throughout the surface of the joint, ex vivousing a multiaxial mechanical tester (Mach-1 v500css, Biomomentum). Thetester records indentation curves on a 64 points grid defined on thecartilage surface prior to the experiment. Indentations were performedperpendicularly at 500 □m/s for a depth of 15 □m. All the measurementwere performed in buffered saline (150 mM NaCl, pH=7.4). Thicknessmapping was performed by replacing the spherical indenter by a 26G ⅜″intradermal bevel needle.

Results

FIG. 34 shows micrographs of the left femoral condyle (left column,control without surgery) and right femoral condyle (right column)treated with either HA or HA+BB. The instantaneous modulus at 15 μm ofthe femoral condyles is also indicated. FIG. 35 compares the loss ofmodulus with treatment with HA or HA+BB. As can be seen, joints treatedwith HA suffered from a reduction of instantaneous compression modulusof more than 80%. Joints those treated with the lubricating fluid of theinvention only demonstrated a loss of instantaneous modulus of less than20% indicating that cartilage degradation rate was significantly loweredwith this treatment.

FIG. 36 shows micrographs of the left femoral condyle (left column,control without surgery) and right femoral condyle (right column)treated with either HA or HA+BB. The mean cartilage thickness over thewhole sample is also indicated. FIG. 37 shows mean cartilage thicknessvariation with treatment with HA or HA+BB compared to CTL.

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

REFERENCES

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety. Thesedocuments include, but are not limited to, the following:

-   Sotcheadt S. et al. J. Orthopaedic Res. 2016 DOI: 10.1002/jor.23330-   X. Banquy et al., Bioinspired bottle-brush polymer exhibits low    friction and amontons-like behavior, J. Am. Chem. Soc.,    136(17):6199-6202, 2014.-   Appleton et al. Arthritis Research & Therapy (2008) 10:407).-   Kaufman et al. Arthritis Research & Therapy (2011) 13:R76.-   (1) Holmberg, K.; Andersson, P.; Erdemir, A. Global Energy    Consumption Due to Friction in Passenger Cars. Tribol. Int. 2012,    47, 221-234.-   (2) Tzanakis, I.; Hadfield, M.; Thomas, B.; Noya, S. M.; Henshaw,    I.; Austen, S. Future Perspectives on Sustainable Tribology.    Renewable Sustainable Energy Rev. 2012, 16, 4126-4140. (3) Moro, T.;    Takatori, Y.; Ishihara, K.; Konno, T.; Takigawa, Y.;-   (3) Matsushita, T.; Chung, U.-i.; Nakamura, K.; Kawaguchi, H.    Surface Grafting of Artificial Joints with a Biocompatible Polymer    for reventing Periprosthetic Osteolysis. Nat. Mater. 2004, 3,    829-836.-   (4) Klein, J. Chemistry. Repair or Replacement—a Joint Perspective.    Science 2009, 323, 47-8.-   (5) Dedinaite, A. Biomimetic Lubrication. Soft Matter 2012, 8,    273284.-   (6) Schmidt, T. A.; Gastelum, N. S.; Nguyen, Q. T.; Schumacher, B.    L.; Sah, R. L. Boundary Lubrication of Articular Cartilage—Role of    Synovial Fluid Constituents. Arthritis Rheum. 2007, 56, 882-891.-   (7) Liu, X.; Dedinaite, A.; Rutland, M.; Thormann, E.; Visnevskij,    C.; Makuska, R.; Claesson, P. M. Electrostatically Anchored Branched    Brush Layers. Langmuir 2012, 28, 15537-47.-   (8) Pettersson, T.; Naderi, A.; Makuska, R.; Claesson, P. M.    Lubrication Properties of Bottle-Brush Polyelectrolytes: An Afm    Study on the Effect of Side Chain and Charge Density. Langmuir 2008,    24, 3336-3347.-   (9) Liu, X.; Thormann, E.; Dedinaite, A.; Rutland, M.; Visnevskij,    C.; Makuska, R.; Claesson, P. M. Low Friction and High Load Bearing    Capacity Layers Formed by Cationic-Block-Non-lonic Bottle-Brush    Copolymers in Aqueous Media. Soft Matter 2013, 9, 5361-5371.-   (10) Chen, M.; Briscoe, W. H.; Armes, S. P.; Cohen, H.; Klein, J.    Polyzwitterionic Brushes: Extreme Lubrication by Design. Eur.    Polym. J. 2011, 47, 511-523.-   (11) Ohsedo, Y.; Takashina, R.; Gong, J. P.; Osada, Y. Surface    Friction of Hydrogels with Well-Defined Polyelectrolyte Brushes.    Langmuir 2004, 20, 6549-6555.-   (12) Raviv, U.; Giasson, S.; Kampf, N.; Gohy, J. F.; Jerome, R.;    Klein, J. Lubrication by Charged Polymers. Nature 2003, 425,    163-165.-   (13) Tairy, O.; Kampf, N.; Driver, M. J.; Armes, S. P.; Klein, J.    Dense, ighly Hydrated Polymer Brushes Via Modified    Atom-TransferRadical-Polymerization: Structure, Surface    Interactions, and Frictional Dissipation. Macromolecules 2015, 48,    140-151.-   (14) Kobayashi, M.; Tanaka, H.; Minn, M.; Sugimura, J.; Takahara, A.    Interferometry Study of Aqueous Lubrication on the Surface of    Polyelectrolyte Brush. ACS Appl. Mater. Interfaces 2014, 6,    2036520371.-   (15) Morse, A. J.; Edmondson, S.; Dupin, D.; Armes, S. P.; Zhang,    Z.; Leggett, G. J.; Thompson, R. L.; Lewis, A. L. Biocompatible    Polymer Brushes Grown from Model Quartz Fibres: Synthesis,    Characterisationand in Situ Determination of Frictional Coefficient.    Soft Matter 2010, 6, 1571-1579.-   (16) Klein, J.; Kumacheva, E.; Mahalu, D.; Perahia, D.;    Fetters, L. J. Reduction of Frictional Forces between Solid-Surfaces    Bearing Polymer Brushes. Nature 1994, 370, 634-636.-   (17) Klein, J. Shear, Friction, and Lubrication Forces between    Polymer-Bearing Surfaces. Annu. Rev. Mater. Sci. 1996, 26, 581-612.-   (18) Banquy, X.; Burdynska, J.; Lee, D. W.; Matyjaszewski, K.;    Israelachvili, J. Bioinspired Bottle-Brush Polymer Exhibits Low    Friction and Amontons-Like Behavior. J. Am. Chem. Soc. 2014, 136,    6199-6202.-   (19) Sheiko, S. S.; Sumerlin, B. S.; Matyjaszewski, K. Cylindrical    Molecular Brushes: Synthesis, Characterization, and Properties.    Prog. Polym. Sci. 2008, 33, 759-785.-   (20) Kobayashi, M.; Terayama, Y.; Kikuchi, M.; Takahara, A. Chain    Dimensions and Surface Characterization of Superhydrophilic Polymer    Brushes with Zwitterion Side Groups. Soft Matter 2013, 9, 5138-5148.-   (21) Chen, M.; Briscoe, W. H.; Armes, S. P.; Klein, J. Lubrication    at Physiological Pressures by Polyzwitterionic Brushes. Science    2009, 323, 1698-1701.-   (22) Maier, G. P.; Rapp, M. V.; Waite, J. H.; Israelachvili, J. N.;    Butler, A. Biological Adhesives. Adaptive Synergy between Catechol    and Lysine Promotes Wet Adhesion by Surface Salt Displacement.    Science 2015, 349, 628-32.-   (23) Petrone, L.; Kumar, A.; Sutanto, C. N.; Patil, N. J.; Kannan,    S.; Palaniappan, A.; Amini, S.; Zappone, B.; Verma, C.; Miserez, A.    Mussel Adhesion Is Dictated by Time-Regulated Secretion and    Molecular Conformation of Mussel Adhesive Proteins. Nat. Commun.    2015, 6, 8737.-   (24) Ma, L.; Gaisinskaya-Kipnis, A.; Kampf, N.; Klein, J. Origins of    Hydration Lubrication. Nat. Commun. 2015, 6, 6060.-   (25) Heuberger, M.; Luengo, G.; Israelachvili, J. Topographic    Information from Multiple Beam Interferometry in the Surface Forces    Apparatus. Langmuir 1997, 13, 3839-3848.-   (26) Perkin, S.; Goldberg, R.; Chai, L.; Kampf, N.; Klein, J.    Dynamic Properties of Confined Hydration Layers. Faraday Discuss.    2009, 141, 399-413.-   (27) Raviv, U.; Perkin, S.; Laurat, P.; Klein, J. Fluidity of Water    Confined Down to Subnanometer Films. Langmuir 2004, 20, 532232.-   (28) Fouviy, S.; Liskiewicz, T.; Kapsa, P.; Hannel, S.; Sauger, E.    An Energy Description of Wear Mechanisms and Its Applications to    Oscillating Sliding Contacts. Wear 2003, 255, 287-298.

What is claimed is:
 1. A method of treating a disease having adegenerative mechanical component, the method comprising administering alubricating fluid to a tissue affected by the disease, wherein thelubricating fluid comprises: a bottle-brush polymer comprising apolymeric backbone with polymeric pendant chains with capping blocksbearing amine groups attached at both ends of said backbone, wherein thepolymeric backbone with polymeric pendant chains form a copolymer offormula:

and a linear polymer which is hyaluronic acid or poly(vinylpyrrolidone)or a pharmaceutically acceptable salt thereof, the bottle-brush polymerand the linear polymer being dissolved together in a solvent.
 2. Themethod of claim 1, wherein the disease is osteoarthritis, a lacrimalfluid production deficiency, or vaginal dryness.
 3. The method of claim2, wherein the disease is osteoarthritis.
 4. The method of claim 1,wherein the lubricating fluid is administered by injection.
 5. Themethod of claim 1, wherein the grafting ratio of the bottle-brushpolymer is between about 40 and about 60%.
 6. The method of claim 1,wherein the linear polymer is partially crosslinked.
 7. The method ofclaim 1, wherein the solvent is saline.
 8. The method of claim 1,wherein the lubricating fluid further comprises one or more therapeuticagent.
 9. A synthetic synovial fluid comprising: a bottle-brush polymercomprising a polymeric backbone with polymeric pendant chains withcapping blocks bearing amine groups attached at both ends of saidbackbone, wherein the polymeric backbone with polymeric pendant chainsform a copolymer of formula:

and a linear polymer which is hyaluronic acid or poly(vinylpyrrolidone)or a pharmaceutically acceptable salt thereof, the bottle-brush polymerand the linear polymer being dissolved together in a solvent.
 10. Thesynthetic synovial fluid of claim 9, being for the treatment ofosteoarthritis.
 11. The synthetic synovial fluid of claim 9, wherein thegrafting ratio of the bottle-brush polymer is between about 40 and about60%.
 12. The synthetic synovial fluid of claim 9, wherein the linearpolymer is partially crosslinked.
 13. The synthetic synovial fluid ofclaim 9, further comprising one or more therapeutic agent.
 14. Thesynthetic synovial fluid of claim 9, wherein the solvent is saline. 15.A method of lubricating a tissue of a living body, the method comprisingthe step of contacting a lubricating fluid with said tissue, wherein thelubricating fluid comprises: a bottle-brush polymer comprising apolymeric backbone with polymeric pendant chains with capping blocksbearing amine groups attached at both ends of said backbone, wherein thepolymeric backbone with polymeric pendant chains form a copolymer offormula:

and a linear polymer which is hyaluronic acid or poly(vinylpyrrolidone)or a pharmaceutically acceptable salt thereof, the bottle-brush polymerand the linear polymer being dissolved together in a solvent.
 16. Themethod of claim 15, wherein the tissue is an eye, skin, a surface of aligament, a vagina, a joint, a gastrointestinal tract, a nasal duct, atracheal duct, or a stomach.
 17. The method of claim 16, wherein thetissue is a joint.
 18. The method of claim 15, wherein the graftingratio of the bottle-brush polymer is between about 40 and about 60%. 19.The method of claim 15, wherein the linear polymer is partiallycrosslinked.
 20. The method of claim 15, wherein the lubricating fluidfurther comprises one or more therapeutic agent.